Polynucleotides for multivalent rna interference, compositions and methods of use thereof

ABSTRACT

The present invention includes bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and/or in which the target regions are complementary to more than one target gene or target sequence. Also included are compositions comprising such nucleic acid molecules and methods of using the same for multivalent RNA interference and the treatment of a variety of diseases and infections.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.14/954,653, filed Nov. 30, 2015, issuing as U.S. Pat. No. 9,957,505,which is a continuation of U.S. patent application Ser. No. 13/375,460,filed Mar. 23, 2012, issued as U.S. Pat. No. 9,200,276, which is anational phase of International Application No. PCT/US10/36962, filedJun. 1, 2010, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/183,011, filed Jun. 1, 2009, whichis incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 8001 US03_SequenceListing.txt. The text file is107 KB, was created Apr. 30, 2018, and is being submitted electronicallyvia EFS-Web.

BACKGROUND Technical Field

The present invention relates generally to precisely structuredpolynucleotide molecules, and methods of using the same for multivalentRNA interference and the treatment of disease.

Description of the Related Art

The phenomenon of gene silencing, or inhibiting the expression of agene, holds significant promise for therapeutic and diagnostic purposes,as well as for the study of gene function itself. Examples of thisphenomenon include antisense technology and dsRNA forms ofposttranscriptional gene silencing (PTGS) which has become popular inthe form of RNA interference (RNAi).

Antisense strategies for gene silencing have attracted much attention inrecent years. The underlying concept is simple yet, in principle,effective: antisense nucleic acids (NA) base pair with a target RNAresulting in inactivation of the targeted RNA. Target RNA recognition byantisense RNA or DNA can be considered a hybridization reaction. Sincethe target is bound through sequence complementarity, this implies thatan appropriate choice of antisense NA should ensure high specificity.Inactivation of the targeted RNA can occur via different pathways,dependent on the nature of the antisense NA (either modified orunmodified DNA or RNA, or a hybrid thereof) and on the properties of thebiological system in which inhibition is to occur.

RNAi based gene suppression is a widely accepted method in which a senseand an antisense RNA form double-stranded RNA (dsRNA), e.g., as a longRNA duplex, a 19-24 nucleotide duplex, or as a short-hairpin dsRNAduplex (shRNA), which is involved in gene modulation by involving enzymeand/or protein complex machinery. The long RNA duplex and the shRNAduplex are pre-cursors that are processed into small interfering RNA(siRNA) by the endoribonuclease described as Dicer. The processed siRNAor directly introduced siRNA is believed to join the protein complexRISC for guidance to a complementary gene, which is cleaved by theRISC/siRNA complex.

However, many problems persist in the development of effective antisenseand RNAi technologies. For example, DNA antisense oligonucleotidesexhibit only short-term effectiveness and are usually toxic at the dosesrequired; similarly, the use of antisense RNAs has also provedineffective due to stability problems. Also, the siRNA used in RNAi hasproven to result in significant off-target suppression due to eitherstrand guiding cleaving complexes potential involvement in endogenousregulatory pathways. Various methods have been employed in attempts toimprove antisense stability by reducing nuclease sensitivity andchemical modifications to siRNA have been utilized. These includemodifying the normal phosphodiester backbone, e.g., usingphosphorothioates or methyl phosphonates, incorporating2′-OMe-nucleotides, using peptide nucleic acids (PNAs and using3′-terminal caps, such as 3′-aminopropyl modifications or 3′-3′ terminallinkages. However, these methods can be expensive and require additionalsteps. In addition, the use of non-naturally occurring nucleotides andmodifications precludes the ability to express the antisense or siRNAsequences in vivo, thereby requiring them to be synthesized andadministered afterwards. Additionally, the siRNA duplex exhibits primaryefficacy to a single gene and off-target to a secondary gene. Thisunintended effect is negative and is not a reliable RNAi multivalence.

Consequently, there remains a need for effective and sustained methodsand compositions for the targeted, direct inhibition of gene function invitro and in vivo, particularly in cells of higher vertebrates,including a single-molecule complex capable of multivalent geneinhibition.

BRIEF SUMMARY

The present invention provides novel compositions and methods, whichinclude precisely structured oligonucleotides that are useful inspecifically regulating gene expression of one or more genessimultaneously when the nucleotide target site sequence of each is notidentical to the other.

In certain embodiments, the present invention includes an isolatedprecisely structured three-stranded polynucleotide complex comprising aregion having a sequence complementary to a target gene or sequence atmultiple sites or complementary to multiple genes at single sites.

In certain embodiments, the present invention includes an isolatedprecisely structured the polynucleotide comprising a region having asequence complementary to a target gene or sequence at multiple sites orcomplementary to multiple genes at single sites; each having partiallyself-complementary regions. In particular embodiments. theoligonucleotide comprises two or more self-complementary regions. Incertain embodiments, the polynucleotides of the present inventioncomprise RNA, DNA, or peptide nucleic acids.

Certain embodiments relate to polynucleotide complexes of at least threeseparate polynucleotides, comprising (a) a first polynucleotidecomprising a target-specific region that is complementary to a firsttarget sequence, a 5′ region, and a 3′ region; (b) a secondpolynucleotide comprising a target-specific region that is complementaryto a second target sequence, a region, and a 3′ region; and (c) a thirdpolynucleotide comprising a null region or a target-specific region thatis complementary to a third target specific, a 5′ region, and a 3′region, wherein each of the target-specific regions of the first,second, and third polynucleotides are complementary to a differenttarget sequence, wherein the 5′ region of the first polynucleotide iscomplementary to the 3′ region of the third polynucleotide, wherein the3′ region of the first polynucleotide is complementary to the 5′ regionof the second polynucleotide, and wherein the 3′ region of the secondpolynucleotide is complementary to the 5′ region of the thirdpolynucleotide, and wherein the three separate polynucleotides hybridizevia their complementary 3′ and 5′ regions to form a polynucleotidecomplex with a first, second, and third-single stranded region, and afirst, second, and third self-complementary region.

In certain embodiments, the first, second, and/or third polynucleotidecomprises about 15-30 nucleotides. In certain embodiments, the first,second, and/or third polynucleotide comprises about 17-25 nucleotides.In certain embodiments, one or more of the self-complementary regionscomprises about 5-10 nucleotide pairs. In certain embodiments, one ormore of the self-complementary regions comprises about 7-8 nucleotidepairs.

In certain embodiments, each of said first, second, and third targetsequences are present in the same gene, cDNA, mRNA, or microRNA. Incertain embodiments, at least two of said first, second, and thirdtarget sequences are present in different genes, cDNAs, mRNAs, ormicroRNAs.

In certain embodiments, all or a portion of the 5′ and/or 3′ region ofeach polynucleotide is also complementary to the target sequence forthat polynucleotide. In certain embodiments, one or more of theself-complementary regions comprises a 3′ overhang.

Certain embodiments relate to self-hybridizing polynucleotide molecules,comprising (a) a first nucleotide sequence comprising a target-specificregion that is complementary to a first target sequence, a 5′ region,and a 3′ region, (b) a second nucleotide sequence comprising atarget-specific region that is complementary to a second targetsequence, a 5′ region, and a 3′ region; and (c) a third nucleotidesequence comprising a null region or a target-specific region that iscomplementary to a third target sequence, a 5′ region, and a 3′ region,wherein the target-specific regions of each of the first, second, andthird nucleotide sequences are complementary to a different targetsequence, wherein the 5′ region of the first nucleotide sequence iscomplementary to the 3′ region of the third nucleotide sequence, whereinthe 3′ region of the first nucleotide sequence is complementary to the5′ region of the second nucleotide sequence, and wherein the 3′ regionof the second nucleotide sequence is complementary to the 5′ region ofthe third nucleotide sequence, and wherein each of the 5′ regionshybridizes to their complementary 3′ regions to form a self-hybridizingpolynucleotide molecule with a first, second, and third single-strandedregion, and a first, second, and third self-complementary region.

In certain embodiments, the first, second, or third polynucleotidesequences comprise about 15-60 nucleotides. In certain embodiments, thetarget-specific region comprises about 15-30 nucleotides. In certainembodiments, one or more of the self-complementary regions comprisesabout 10-54 nucleotides. In certain embodiments, one or more of theself-complementary regions comprises a 3′ overhang. In certainembodiments, one or more of the self-complementary regions forms astem-loop structure. In certain embodiments, one or more of theself-complementary regions comprises a proximal box of dinucleotidesAG/UU that is outside of the target specific region. In certainembodiments, one or more of the self-complementary regions comprises adistal box of 4 nucleotides that is outside of the target-specificregion, wherein the third nucleotide of the distal box is not a G. Alsoincluded are vectors that encode a self-hybridizing polynucleotidemolecule, as described herein.

In certain embodiments, each of said first, second, and third targetsequences are present in the same gene, cDNA, mRNA, or microRNA. Incertain embodiments, at least two of said first, second, and thirdtarget sequences are present in different genes, cDNAs, mRNAs, ormicroRNAs.

In certain embodiments, a self-complementary region comprises astem-loop structure composed of a bi-loop, tetraloop or larger loop. Incertain embodiments, the sequence complementary to a target genesequence comprises at least 17 nucleotides, or 17 to 30 nucleotides,including all integers in between.

In certain embodiments, the self-complementary region (ordouble-stranded region) comprises at least 5 nucleotides, at least 6nucleotides, at least 24 nucleotides, or 12 to 54 or 60 nucleotides,including all integers in between.

In certain embodiments, a loop region of a stem-loop structure comprisesat least 1 nucleotide. In certain embodiments, the loop region comprisesat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,or at least 8 nucleotides.

In further embodiments, a loop region of a stem-loop structure iscomprised of a specific tetra-loop sequence NGNN or AAGU or UUUU or UUGAor GUUA, where these sequences are 5′ to 3′.

In a further embodiment, the present invention includes an expressionvector capable of expressing a polynucleotide of the present invention.In various embodiments, the expression vector is a constitutive or aninducible vector.

The present invention further includes a composition comprising aphysiologically acceptable carrier and a polynucleotide of the presentinvention.

In other embodiments, the present invention provides a method forreducing the expression of a gene, comprising introducing apolynucleotide complex or molecule of the present invention into a cell.In various embodiments, the cell is plant, animal, protozoan, viral,bacterial, or fungal. In one embodiment, the cell is mammalian.

In some embodiments, the polynucleotide complex or molecule isintroduced directly into the cell, while in other embodiments, thepolynucleotide complex or molecule is introduced extracellularly by ameans sufficient to deliver the isolated polynucleotide into the cell.

In another embodiment, the present invention includes a method fortreating a disease, comprising introducing a polynucleotide complex ormolecule of the present invention into a cell, wherein overexpression ofthe targeted gene is associated with the disease. In one embodiment, thedisease is a cancer.

The present invention further provides a method of treating an infectionin a patient, comprising introducing into the patient a polynucleotidecomplex or molecule of the present invention, wherein the isolatedpolynucleotide mediates entry, replication, integration, transmission,or maintenance of an infective agent.

In yet another related embodiment, the present invention provides amethod for identifying a function of a gene, comprising introducing intoa cell a polynucleotide complex or molecule of the present invention,wherein the polynucleotide complex or molecule inhibits expression ofthe gene, and determining the effect of the introduction of thepolynucleotide complex or molecule on a characteristic of the cell,thereby determining the function of the targeted gene. In oneembodiment, the method is performed using high throughput screening.

In a further embodiment, the present invention provides a method ofdesigning a polynucleotide sequence comprising two or moreself-complementary regions for the regulation of expression of a targetgene, comprising: (a) selecting the first three guide sequences 17 to 25nucleotides in length and complementary to a target gene or multipletarget genes; (b) selecting one or more additional sequences 4 to 54nucleotides in length, which comprises self-complementary regions andwhich are not fully-complementary to the first sequence; and optionally(c) defining the sequence motif in (b) to be complementary,non-complementary, or replicate a gene sequence which arenon-complementary to the sequence selected in step (a).

In another embodiment, the mutated gene is a gene expressed from a geneencoding a mutant p53 polypeptide. In another embodiment, the gene isviral, and may include one or more different viral genes. In specificembodiments, the gene is an HIV gene, such as gag, pol, env, or tat,among others described herein and known in the art. In otherembodiments, the gene is ApoB.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 6 illustrate exemplary polynucleotide structures of thepresent invention.

FIG. 1 shows a polynucleotide complex of three separate polynucleotidemolecules. (A) indicates the region comprising sequence complementary toa site on a target gene (hatched); (B) indicates the region comprisingsequence complementary to a second site on the target gene or a site ona different gene (cross-hatched); (C) indicates the region comprisingsequence complementary to a third site on the target gene or a site on adifferent gene (filled in black). The numbers 1, 2, and 3 indicate the3′ end of each oligonucleotide that guides gene silencing; (A) loads inthe direction of 1, (B) in the direction 2, and (C) in the direction 3.The 3′ and 5′ regions of each molecule, which hybridize to each other toform their respective self-complementary or double-stranded regions, areindicated by connecting bars. Each polynucleotide comprises a twonucleotide 3′ overhang.

FIG. 2 shows a single, self-hybridizing polynucleotide of the invention,having three single-stranded regions and three self-complementaryregions, which is a precursor for processing into a core molecule. Thetarget specific regions are darkened. (D) indicates a self-complementarystem-loop region (filled in white) capped with a tetraloop of fournucleotides; (D) also indicates a stem-loop region having a 14/16nucleotide cleavage site within the stem-loop structure; cleavage mayoccur by RNase III to remove the stem loop nucleotides shown in white);(E) indicates a distal box wherein the third nucleotide as determined 5′to 3′ is not a G, since it is believed that the presence of a G wouldblock RNase III cleavage required for removal of the stem-loop region;(F) indicates a proximal box of dinucleotides AG/UU, which is an in vivodeterminant of RNase III recognition and binding of RNase III (Nichols2000); (G) indicates a tetraloop. The polynucleotide molecule shown inFIG. 2 is a longer transcript RNA that is ‘pre-processed’ in the cell byRNase III. The resulting RNA structure is identical to the structuredepicted in FIG. 1.

FIG. 3 depicts a self-forming single-stranded oligonucleotide withtetraloop formats. (H) indicates a tetraloop; (I) indicates a tri-loopconnecting two core strands when the leading strand incorporates a 2nucleotide overhang. In this structure, tetraloops are used to mimicwhat would be a 3′ hydroxyl/5′ phosphate of the overhangs in thestructure shown in FIG. 1 and function more directly than those of thestructure shown in FIG. 2. As demonstrated in Example 2, this shorttetraloop format guides silencing directly without pre-processing. It isbelieved that the GUUA loop twists the nucleotides in the loop andexpose the hydrogens (see, e.g., Nucleic Acids Research, 2003, Vol. 31No. 3, FIG. 6, page 1094). This structure is compatible with PAZ orRISC.

FIG. 4 depicts a self-forming single stranded oligonucleotide fordivalent use. (J) indicates a larger loop connecting two core strands;(K) indicates the key strand as completing the complex formation but“null” to a target gene, i.e., not-specific for a target gene. The twotarget specific regions are shaded. This structure is a composition for‘divalent’ use when working with RNA transcripts. Since chemicalmodifications are not possible, the structure determines asymmetricallyof loading and silencing activity. The first 19 nucleotides of themolecule is the PRIMARY strand, (K) indicates a KEY strand that isdeactivated, and the SECONDARY strand is the last 21 nucleotides of themolecule. The first priority of loading into RISC and functioning is theSECONDARY strand by exposed 5′/3′ ends. The next priority is the PRIMARYstrand, which is exposed after RNase III pre-processing in the cell. The3′ end of the nullified KEY strand is not functional, since the largeloop is not processed nor is compatible with loading into RISC itself.

FIG. 5 depicts a polynucleotide complex of the present invention havingmodified RNA bases. (L), (M), and (N) illustrate regions (defined byhashed lines) in which the Tm can be incrementally increased by the useof modified RNA (e.g., 2′-O-methyl RNA or 2′-fluoro RNA instead of 2′-OHRNA) to preference the annealing and/or the silencing order of ends 1, 2or 3.

FIG. 6 depicts two embodiments of oligonucleotide complexes of thepresent invention. (O) illustrates a blunt-ended DNA strand thatdeactivates the silencing function of this strand; and (P) illustratesan end that can be utilized for conjugation of a delivery chemistry,ligand, antibody, or other payload or targeting molecule.

FIGS. 7A and 7B show the results of suppression of GFP expression bymultivalent-siRNA molecules of the invention, as compared to standardshRNA molecules (see Example 1). FIG. 7A shows increased suppression ofGFP by MV clone long I (108%) and MV clone long II (119%), relative toshRNA control (set at 100%). FIG. 7B shows increased suppression of GFPexpression by synthetic MV-siRNA GFP I (127%), relative to shRNA control(set at 100%), which is slightly reduced when one of the strands of thesynthetic MV-siRNA complex is replaced by a DNA strand (MF-siRNA GFP IDNA (116%)).

FIGS. 8A, 8B and 8C show exemplary targeting regions (underlined) forthe GFP coding sequence (SEQ ID NO:8). FIG. 8A shows the regions thatwere targeted by the MV-siRNA molecules of Tables 1 and 2 in Example 1.FIGS. 8B and 8C show additional exemplary targeting regions.

FIG. 9 shows the inhibitory effects of MV-siRNA molecules on HIVreplication, in which a di-valent MV-siRNA targeted to both gag and tathas a significantly greater inhibitory effect on HIV replication than ansiRNA targeted to gag only. The di-valent MV-siRNA exhibited 56.89%inhibition at 10 days and 60.02% inhibition at 40 days, as compared tothe siRNA targeted to gag alone, which exhibited 19.77% inhibition at 10days and 32.43% inhibition at 40 days.

FIGS. 10A, 10B, 10C and 10D show the nucleotide sequence of an exemplaryHIV genome (SEQ ID NO:9), which can be targeted according to theMV-siRNA molecules of the present invention. This sequence extends fromFIG. 10A through FIG. 10D.

FIG. 11 shows the nucleotide sequence of the env gene (SEQ ID NO:4),derived from the HIV genomic sequence of FIG. 10.

FIGS. 12A and 12B provide additional HIV sequences. FIG. 12A shows thenucleotide sequence of the gag gene (SEQ ID NO:2), and FIG. 12B showsthe nucleotide sequence of the tat gene (SEQ ID NO:3), both of which arederived from the HIV genomic sequence of FIG. 10.

FIGS. 13A, 13B, 13C, 13D and 13E show the coding sequence of murineapolipoprotein B (ApoB) (SEQ ID NO:10), which can be targeted usingcertain MV-siRNAs provided herein. This sequence extends from FIG. 13Athrough FIG. 13E.

FIGS. 14A, 14B, 14C, 14D and 14E show the mRNA sequence of humanapolipoprotein B (apoB) (SEQ ID NO:1), which can be targeted usingcertain MV-siRNAs provided herein. This sequence extends from FIG. 14Athrough FIG. 14E.

DETAILED DESCRIPTION

The present invention provides novel compositions and methods forinhibiting the expression of a gene at multiple target sites, or forinhibiting the expression of multiple genes at one or more target sites,which sites are not of equivalent nucleotide sequences, in eukaryotes invivo and in vitro. In particular, the present invention providespolynucleotide complexes and polynucleotide molecules comprising two,three, or more regions having sequences complementary to regions of oneor more target genes, which are capable of targeting and reducingexpression of the target genes. In various embodiments, the compositionsand methods of the present invention may be used to inhibit theexpression of a single target gene by targeting multiple sites withinthe target gene or its expressed RNA. Alternatively, they may be used totarget two or more different genes by targeting sites within two or moredifferent genes or their expressed RNAs.

The present invention offers significant advantages over traditionalsiRNA molecules. First, when polynucleotide complexes or molecules ofthe present invention target two or more regions within a single target,gene, they are capable of achieving greater inhibition of geneexpression from the target gene, as compared to an RNAi agent thattargets only one region within a target gene. In addition,polynucleotide complexes or molecules of the present invention thattarget two or more different target genes may be used to inhibit theexpression of multiple target genes associated with a disease ordisorder using a single polynucleotide complex or molecule. Furthermore,polynucleotide complexes and molecules of the present invention do notrequire the additional non-targeting strand present in conventionaldouble-stranded RNAi agents, so they do not have off-target effectscaused by the non-targeting strand. Accordingly, the polynucleotidecomplexes and molecules of the present invention offer surprisingadvantages over polynucleotide inhibitors of the prior art, includingantisense RNA and RNA interference molecules, including increasedpotency and increased effectiveness against one or more target genes.

The present invention is also based upon the recognition of thepolynucleotide structure guiding a protein complex for cleavage usingonly one, two, or three of the guide strands, which are complementary toone, two, or three distinct nucleic sequences of the target genes. Thismultivalent function results in a markedly broader and potent inhibitionof a target gene or group of target genes than that of dsRNA, whileutilizing many of the same endogenous mechanisms.

Certain embodiments of the present invention are also based upon therecognition of the polynucleotide structure directionally bypresentation of the 3′ overhangs and 5′ phosphate resulting in a sensestrand free complex, which contributes to greater specificity than thatof dsRNA-based siRNA.

Given their effectiveness, the compositions of the present invention maybe delivered to a cell or subject with an accompanying guarantee ofspecificity predicted by the single guide strand complementary to thetarget gene or multiple target genes.

Multivalent siRNAs

The present invention includes polynucleotide complexes and moleculesthat comprise two or more targeting regions complementary to regions ofone or more target genes. The polynucleotide complexes and molecules ofthe present invention may be referred to as multivalent siRNAs(mv-siRNAs), since they comprise at least two targeting regionscomplementary to regions of one or more target genes. Accordingly, thecompositions and methods of the present invention may be used to inhibitor reduce expression of one or more target genes, either by targetingtwo or more regions within a single target gene, or by targeting one ormore regions within two or more target genes.

In certain embodiments, polynucleotide complexes of the presentinvention comprise three or more separate oligonucleotides, each havinga 5′ and 3′ end, with two or more of the oligonucleotides comprising atargeting region, which oligonucleotides hybridize to each other asdescribed herein to form a complex. Each of the strands is referred toherein as a “guide strand.” In other embodiments, polynucleotidemolecules of the present invention are a single polynucleotide thatcomprises three or more guide strands, with two or more of the guidestrands comprising a targeting region, which polynucleotide hybridizesto itself through self-complementary regions to form a structuredescribed herein. The resulting structure may then be processed, e.g.,intracellularly, to remove loop structures connecting the various guidestrands. Each guide strand, which may be present in differentoligonucleotides or within a single polynucleotide, comprises regionscomplementary to other guide strands.

In certain embodiments, the present invention provides polynucleotidecomplexes and molecules that comprise at least three guide strands, atleast two of which comprise regions that are complementary to differentsequences within one or more target genes. In various embodiments, thepolynucleotide complexes of the present invention comprise two, three ormore separate polynucleotides each comprising one or more guide strands,which can hybridize to each other to form a complex. In otherembodiments, the polynucleotide molecules of the present inventioncomprise a single polynucleotide that comprises three or more guidestrands within different regions of the single polynucleotide.

Certain embodiments of the present invention are directed topolynucleotide complexes or molecules having at least three guidestrands, two or more of which are partially or fully complementary toone or more target genes; and each having about 4 to about 12, about 5to about 10, or preferably about 7 to about 8, nucleotides on either endthat are complementary to each other (i.e., complementary to a region ofanother guide strand), allowing the formation of a polynucleotidecomplex (see, e.g., FIG. 1). For example, each end of a guide strand maycomprise nucleotides that are complementary to nucleotides at one end ofanother of the guide strands of the polynucleotide complex or molecule.Certain embodiments may include polynucleotide complexes that comprise4, 5, 6 or more individual polynucleotide molecules or guide strands.

In certain embodiments, a polynucleotide complex of the presentinvention comprises at least three separate polynucleotides, whichinclude: (1) a first polynucleotide comprising a target-specific regionthat is complementary to a first target sequence, a 5′ region, and a 3′region; (2) a second polynucleotide comprising a target-specific regionthat is complementary to a second target sequence, a 5′ region, and a 3′region; and (3) a third polynucleotide comprising either a null regionor a target-specific region that is complementary to a third targetspecific, a 5′ region, and a 3′ region, wherein each of thetarget-specific regions of the first, second, and third polynucleotidesare complementary to a different target sequence, wherein the 5′ regionof the first polynucleotide is complementary to the 3′ region of thethird polynucleotide, wherein the 3′ region of the first polynucleotideis complementary to the 5′ region of the second polynucleotide, andwherein the 3′ region of the second polynucleotide is complementary tothe 5′ region of the third polynucleotide, and wherein the threeseparate polynucleotides hybridize via their complementary 3′ and 5′regions to form a polynucleotide complex with a first, second, and thirdsingle-stranded region, and a first, second, and thirdself-complementary region.

As described above, in particular embodiments, a polynucleotide complexof the present invention comprises at least three separateoligonucleotides, each having a 5′ end and a 3′ end. As depicted in FIG.1, a region at the 5′ end of the first oligonucleotide anneals to aregion at the 3′ end of the third oligonucleotide; a region at the 5′end of the third oligonucleotide anneals to a region at the 3′ end ofthe second oligonucleotide; and a region at the 5′ end of the secondoligonucleotide anneals to a region at the 3′ end of the firstoligonucleotide. If additional oligonucleotides are present in thecomplex, then they anneal to other oligonucleotides of the complex in asimilar manner. The regions at the ends of the oligonucleotides thatanneal to each other may include the ultimate nucleotides at either orboth the 5′ and/or 3′ ends. Where the regions of both the hybridizing 3′and 5′ ends include the ultimate nucleotides of the oligonucleotides,the resulting double-stranded region is blunt-ended. In particularembodiments, the region at the 3′ end that anneals does not include theultimate and/or penultimate nucleotides, resulting in a double-strandedregion having a one or two nucleotide 3′ overhang.

In certain embodiments, the guide strands are present in a singlepolynucleotide molecule, and hybridize to form a single,self-hybridizing polynucleotide with three single-stranded regions andthree self-complementary regions (or double-stranded regions), and atleast two target-specific regions (see, e.g., FIG. 2). In relatedembodiments, a single molecule may comprise at least 3, at least 4, atleast 5 or at least 6 guide strands, and forms a single,self-hybridizing polynucleotide with at least 3, at least 4, at least 5,or at least 6 self-complementary regions (or double-stranded regions),and at least 2, at least 3, at least 4, or at least 5 target-specificregions, respectively. In particular embodiments, this single,self-hybridizing polynucleotide is a precursor molecule that may beprocessed by the cell to remove the loop regions and, optionally, anamount of proximal double-stranded region, resulting in an activemv-siRNA molecule (see, e.g., FIG. 2).

Thus, in particular embodiments, the present invention includes aself-hybridizing polynucleotide molecule, comprising: (1) a firstnucleotide sequence comprising a target-specific region that iscomplementary to a first target sequence, a 5′ region, and a 3′ region,(2) a second nucleotide sequence comprising a target-specific regionthat is complementary to a second target sequence, a 5′ region, and a 3′region; and (3) a third nucleotide sequence comprising a null region ofa target-specific region that is complementary to a third targetsequence, a 5′ region, and a 3′ region, wherein the target-specificregions of each of the first, second, and third nucleotide sequences arecomplementary to a different target sequence, wherein the 5′ region ofthe first nucleotide sequence is complementary to the 3′ region of thethird nucleotide sequence, wherein the 3′ region of the first nucleotidesequence is complementary to the 5′ region of the second nucleotidesequence, and wherein the 3′ region of the second nucleotide sequence iscomplementary to the 5′ region of the third nucleotide sequence, andwherein each of the 5′ regions hybridizes to their complementary 3′regions to form a self-hybridizing polynucleotide molecule with a first,second, and third single-stranded region, and a first, second, and thirdself-complementary region.

In particular embodiments, a single, self-hybridizing polynucleotide ofthe present invention may comprise one or more cleavable nucleotides inthe single-stranded loops that form when the polynucleotide is annealedto itself. Once the single, self-hybridizing polynucleotide is annealedto itself, the cleavable nucleotides may be cleaved to result in apolynucleotide complex comprising three or more separateoligonucleotides. Examples of cleavable nucleotides that may be usedaccording to the present invention include, but are not limited to,photocleavable nucleotides, such as pcSpacer (Glen Research Products,Sterling, Va., USA), or phosphoramadite nucleotides.

As used herein, polynucleotides complexes and molecules of the presentinvention include isolated polynucleotides comprising threesingle-stranded regions, at least two of which are complementary to twoor more target sequences, each target sequence located within one ormore target genes, and comprising at least two or threeself-complementary regions interconnecting the 5′ or 3′ ends of thesingle-stranded regions, by forming a double-stranded region, such as astem-loop structure. The polynucleotides may also be referred to hereinas the oligonucleotides.

In certain embodiments, the polynucleotide complexes and molecules ofthe present invention comprise two or more regions of sequencecomplementary to a target gene. In particular embodiments, these regionsare complementary to the same target genes or genes, while in otherembodiments, they are complementary to two or more different targetgenes or genes.

Accordingly, the present invention includes one or moreself-complementary polynucleotides that comprise a series of sequencescomplementary to one or more target genes or genes. In particularembodiments, these sequences are separated by regions of sequence thatare non-complementary or semi-complementary to a target gene sequenceand non-complementary to a self-complementary region. In otherembodiments of the polynucleotide comprising multiple sequences that arecomplementary to target genes or genes, the polynucleotide comprises aself-complementary region at the 5′ end, 3 end′, or both ends of one ormore regions of sequence complementary to a target gene. In a particularembodiment, a polynucleotide comprises two or more regions of sequencecomplementary to one or more target genes, with self-complementaryregions located at the 5′ and 3′ end of each guide strand that iscomplementary to a target gene. In certain embodiments, all or a portionof these 3′ and 5′ regions may be complementary to the target sequence,in addition to being complementary to their corresponding 3′ or 5′regions.

The term “complementary” refers to nucleotide sequences that are fullyor partially complementary to each other, according to standard basepairing rules. The term “partially complementary” refers to sequencesthat have less than full complementarity, but still have a sufficientnumber of complementary nucleotide pairs to support binding orhybridization within the stretch of nucleotides under physiologicalconditions.

In particular embodiments, the region of a guide strand complementary toa target gene (i.e., the targeting region) may comprise one or morenucleotide mismatches as compared to the target gene. Optionally, themismatched nucleotide(s) in the guide strand may be substituted with anunlocked (UNA) nucleic acid or a phosphoramidite nucleic acid (e.g.,rSpacer, Glen Research, Sterling, Va., USA), to allow base-pairing,e.g., Watson-Crick base pairing, of the mismatched nucleotide(s) to thetarget gene.

As used herein, the term “self-complementary” or “self-complementaryregion” may refer to a region of a polynucleotide molecule of theinvention that binds or hybridizes to another region of the samemolecule to form A-T(U) and G-C hybridization pairs, thereby forming adouble stranded region; and/or it may refer to a region of a firstnucleotide molecule that binds to a region of a second or thirdnucleotide molecule to form a polynucleotide complex of the invention(i.e., an RNAi polynucleotide complex), wherein the complex is capableof RNAi interference activity against two or more target sites. The tworegions that bind to each other to form the self-complementary regionmay be contiguous or may be separated by other nucleotides. Also, as inan RNAi polynucleotide complex, the two regions may be on separatenucleotide molecules.

In certain embodiments, a “self-complementary region” comprises a “3′region” of a first defined nucleotide sequence that is bound orhybridized to a “5′ region” of a second or third defined nucleotidesequence, wherein the second or third defined sequence is within thesame molecule—to form a self-hybridizing polynucleotide molecule. Incertain embodiments, a “self-complementary region” comprises a “3′region” of a first polynucleotide molecule that is bound or hybridizedto a “5′ region” of a separate polynucleotide molecule, to form apolynucleotide complex. These 3′ and 5′ regions are typically defined inrelation to their respective target-specific region, in that the 5′regions are on the 5′ end of the target-specific region and the 3′regions are on the 3′ end of the target specific region. In certainembodiments, one or both of these 3′ and 5′ regions not only hybridizeto their corresponding 3′ or 5′ regions to form a self-complementaryregion, but may be designed to also contain full or partialcomplementarily their respective target sequence, thereby forming partof the target-specific region. In these embodiments, the target-specificregion contains both a single-stranded region and self-complementary(i.e., double-stranded) region.

In certain embodiments, these “self-complementary regions” compriseabout 5-12 nucleotide pairs, preferably 5-10 or 7-8 nucleotide pairs,including all integers in between. Likewise, in certain embodiments,each 3′ region or 5′ region comprises about 5-12 nucleotides, preferably5-10 or 7-8 nucleotides, including all integers in between.

The term “non-complementary” indicates that in a particular stretch ofnucleotides, there are no nucleotides within that align with a target toform A-T(U) or G-C hybridizations. The term “semi-complementary”indicates that in a stretch of nucleotides, there is at least onenucleotide pair that aligns with a target to form an A-T(U) or G-Chybridizations, but there are not a sufficient number of complementarynucleotide pairs to support binding within the stretch of nucleotidesunder physiological conditions.

The term “isolated” refers to a material that is at least partially freefrom components that normally accompany the material in the material'snative state. Isolation connotes a degree of separation from an originalsource or surroundings. Isolated, as used herein, e.g., related to DNA,refers to a polynucleotide that is substantially away from other codingor non-coding sequences, and that the DNA molecule can contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

In various embodiments, a polynucleotide complex or molecule of thepresent invention comprises RNA, DNA, or peptide nucleic acids, or acombination of any or all of these types of molecules. In addition, apolynucleotide may comprise modified nucleic acids, or derivatives oranalogs of nucleic acids. General examples of nucleic acid modificationsinclude, but are not limited to, biotin labeling, fluorescent labeling,amino modifiers introducing a primary amine into the polynucleotide,phosphate groups, deoxyuridine, halogenated nucleosides,phosphorothioates, 2′-O-Methyl RNA analogs, chimeric RNA analogs, wobblegroups, universal bases, and deoxyinosine.

A “subunit” of a polynucleotide or oligonucleotide refers to onenucleotide (or nucleotide analog) unit. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g., a phosphate orphosphorothioate linkage or a cationic linkage). A given syntheticMV-siRNA may utilize one or more different types of subunits and/orintersubunit linkages, mainly to alter its stability, Tm, RNasesensitivity, or other characteristics, as desired. For instance, certainembodiments may employ RNA subunits with one or more 2′-O-methyl RNAsubunits.

The cyclic subunits of a polynucleotide or an oligonucleotide may bebased on ribose or another pentose sugar or, in certain embodiments,alternate or modified groups. Examples of modified oligonucleotidebackbones include, without limitation, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Alsocontemplated are peptide nucleic acids (PNAs), locked nucleic acids(LNAs), 2′-O-methyl oligonucleotides (2′-OMe), 2′-methoxyethoxyoligonucleotides (MOE), among other oligonucleotides known in the art.

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses or chemistries of theoligomers, Ts and Us are interchangeable. For instance, with otherantisense chemistries such as 2′-O-methyl antisense oligonucleotidesthat are more RNA-like, the T bases may be shown as U.

As noted above, certain polynucleotides or oligonucleotides providedherein include one or more peptide nucleic acid (PNAs) subunits. Peptidenucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm, Buchardtet al. 1993). The backbone of PNAs is formed by peptide bonds ratherthan phosphodiester bonds, making them well-suited for antisenseapplications (see structure below). A backbone made entirely of PNAs isuncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greaterthan normal thermal stability. PNAs are not recognized by nucleases orproteases.

PNAs may be produced synthetically using any technique known in the art.PNA is a DNA analog in which a polyamide backbone replaces thetraditional phosphate ribose ring of DNA. Despite a radical structuralchange to the natural structure, PNA is capable of sequence-specificbinding in a helix form to DNA or RNA. Characteristics of PNA include ahigh binding affinity to complementary DNA or RNA, a destabilizingeffect caused by single-base mismatch, resistance to nucleases andproteases, hybridization with DNA or RNA independent of saltconcentration and triplex formation with homopurine DNA. Panagene™ hasdeveloped its proprietary Bts PNA monomers (Bts;benzothiazole-2-sulfonyl group) and proprietary oligomerisation process.The PNA oligomerisation using Bts PNA monomers is composed of repetitivecycles of deprotection, coupling and capping. Panagene's patents to thistechnology include U.S. Pat. No. 6,969,766, U.S. Pat. No. 7,211,668,U.S. Pat. No. 7,022,851, U.S. Pat. No. 7,125,994, U.S. Pat. No.7,145,006 and U.S. Pat. No. 7,179,896. Representative United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al. Science, 1991, 254, 1497.

Also included are “locked nucleic acid” subunits (LNAs). The structuresof LNAs are known in the art: for example, Wengel, et al., ChemicalCommunications (1998) 455; Tetrahedron (1998) 54, 3607, and Accounts ofChem. Research (1999) 32, 301); Obika, et al., Tetrahedron Letters(1997) 38, 8735; (1998) 39, 5401, and Bioorganic Medicinal Chemistry(2008)16, 9230.

Polynucleotides and oligonucleotides may incorporate one or more LNAs;in some cases, the compounds may be entirely composed of LNAs. Methodsfor the synthesis of individual LNA nucleoside subunits and theirincorporation into oligonucleotides are known in the art: U.S. Pat. Nos.7,572,582; 7,569,575; 7,084,125; 7,060,809; 7,053,207; 7,034,133;6,794,499; and 6,670,461. Typical intersubunit linkers includephosphodiester and phosphorothioate moieties; alternatively,non-phosphorous containing linkers may be employed. One embodimentincludes an LNA containing compound where each LNA subunit is separatedby a RNA or a DNA subunit (i.e., a deoxyribose nucleotide). Furtherexemplary compounds may be composed of alternating LNA and RNA or DNAsubunits where the intersubunit linker is phosphorothioate.

Certain polynucleotides or oligonucleotides may comprisemorpholino-based subunits bearing base-pairing moieties, joined byuncharged or substantially uncharged linkages. The terms “morpholinooligomer” or “PMO” (phosphoramidate- or phosphorodiamidate morpholinooligomer) refer to an oligonucleotide analog composed of morpholinosubunit structures, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, preferably twoatoms long, and preferably uncharged or cationic, joining the morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,and (ii) each morpholino ring bears a purine or pyrimidine or anequivalent base-pairing moiety effective to bind, by base specifichydrogen bonding, to a base in a polynucleotide.

Variations can be made to this linkage as long as they do not interferewith binding or activity. For example, the oxygen attached to phosphorusmay be substituted with sulfur (thiophosphorodiamidate). The 5′ oxygenmay be substituted with amino or lower alkyl substituted amino. Thependant nitrogen attached to phosphorus may be unsubstituted,monosubstituted, or disubstituted with (optionally substituted) loweralkyl. The purine or pyrimidine base pairing moiety is typicallyadenine, cytosine, guanine, uracil, thymine or inosine. The synthesis,structures, and binding characteristics of morpholino oligomers aredetailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337, and PCT Appn. Nos. PCT/US07/11435(cationic linkages) and U.S. Ser. No. 08/012,804 (improved synthesis),all of which are incorporated herein by reference.

In one aspect of the invention, MV-siRNA comprise at least one ligandtethered to an altered or non-natural nucleobase. Included are payloadmolecules and targeting molecules. A large number of compounds canfunction as the altered base. The structure of the altered base isimportant to the extent that the altered base should not substantiallyprevent binding of the oligonucleotide to its target, e.g., mRNA. Incertain embodiments, the altered base is difluorotolyl, nitropyrrolyl,nitroimidazolyl, nitroindolyl, napthalenyl, anthrancenyl, pyridinyl,quinolinyl, pyrenyl, or the divalent radical of any one of thenon-natural nucleobases described herein. In certain embodiments, thenon-natural nucleobase is difluorotolyl, nitropyrrolyl, ornitroimidazolyl. In certain embodiments, the non-natural nucleobase isdifluorotolyl.

A wide variety ligands are known in the art and are amenable to thepresent invention. For example, the ligand can be a steroid, bile acid,lipid, folic acid, pyridoxal, B12, riboflavin, biotin, aromaticcompound, polycyclic compound, crown ether, intercalator, cleavermolecule, protein-binding agent, or carbohydrate. In certainembodiments, the ligand is a steroid or aromatic compound. In certaininstances, the ligand is cholesteryl.

In other embodiments, the polynucleotide or oligonucleotide is tetheredto a ligand for the purposes of improving cellular targeting and uptake.For example, an MV-siRNA agent may be tethered to an antibody, orantigen binding fragment thereof. As an additional example, an MV-siRNAagent may be tethered to a specific ligand binding molecule, such as apolypeptide or polypeptide fragment that specifically binds a particularcell-surface receptor, or that more generally enhances cellular uptake,such as an arginine-rich peptide.

The term “analog” as used herein refers to a molecule, compound orcomposition that retains the same structure and/or function (e.g.,binding to a target) as a polynucleotide herein. Examples of analogsinclude peptidomimetic and small and large organic or inorganiccompounds.

The term “derivative” or “variant” as used herein refers to apolynucleotide that differs from a naturally occurring polynucleotide(e.g., target gene sequence) by one or more nucleic acid deletions,additions, substitutions or side-chain modifications. In certainembodiments, variants have at least 70%, at least 80% at least 90%, atleast 95%, or at least 99% sequence identity to a region of a targetgene sequence. Thus, for example, in certain embodiments, anoligonucleotide of the present invention comprises a region that iscomplementary to a variant of a target gene sequence.

Polynucleotide complexes and molecules of the present invention comprisea sequence region, or two or more sequence regions, each of which iscomplementary, and in particular embodiments completely complementary,to a region of a target gene or polynucleotide sequences (or a variantthereof). In particular embodiments, a target gene is a mammalian gene,e.g., a human gene, or a gene of a microorganism infecting a mammal,such as a virus. In certain embodiments, a target gene is a therapeutictarget. For example, a target gene may be a gene whose expression oroverexpression is associated with a human disease or disorder. This maybe a mutant gene or a wild type or normal gene. A variety of therapeutictarget genes have been identified, and any of these may be targeted bypolynucleotide complexes and molecules of the present invention.Therapeutic target genes include, but are not limited to, oncogenes,growth factor genes, translocations associated with disease such asleukemias, inflammatory protein genes, transcription factor genes,growth factor receptor genes, anti-apoptotic genes, interleukins, sodiumchannel genes, potassium channel genes, such as, but not limited to thefollowing genes or genes encoding the following proteins: apolipoproteinB (ApoB), apolipoprotein B-100 (ApoB-100), bcl family members, includingbcl-2 and bcl-x, MLL-AF4, Huntington gene, AML-MT68 fusion gene, IKK-B,Aha1, PCSK9, Eg5, transforming growth factor beta (TGFbeta), Nav1.8,RhoA, HIF-1alpha, Nogo-L, Nogo-R, toll-like receptor 9 (TLR9), vascularendothelial growth factor (VEGF), SNCA, beta-catenin, CCR5, c-myc, p53,interleukin-1, interleukin 2, interleukin-12, interleukin-6,interleukin-17a (IL-17a), interleukin-17f (IL-17f), Osteopontin (OPN)gene, psoriasis gene, and tumor necrosis factor gene.

In particular embodiments, polynucleotide complexes or molecules of thepresent invention comprise guide strands or target-specific regionstargeting two or more genes, e.g., two or more genes associated with aparticular disease or disorder. For example, they may include guidestrands complementary to interleukin-1 gene or mRNA and tumor necrosisfactor gene or mRNA; complementary to interleukin-1 gene or mRNA andinterleukin-12 gene or mRNA; or complementary to interleukin-1 gene ormRNA, interleukin-12 gene or mRNA and tumor necrosis factor gene ormRNA, for treatment of rheumatoid arthritis. In one embodiment, theyinclude guide strands complementary to osteopontin gene or mRNA and TNFgene or mRNA.

Other examples of therapeutic target genes include genes and mRNAsencoding viral proteins, such as human immunodeficiency virus (HIV)proteins, HTLV virus proteins, hepatitis C virus (HCV) proteins, Ebolavirus proteins, JC virus proteins, herpes virus proteins, human polyomavirus proteins, influenza virus proteins, and Rous sarcoma virusproteins. In particular embodiments, polynucleotide complexes ormolecules of the present invention include guide strands complementaryto two or more genes or mRNAs expressed by a particular virus, e.g., twoor more HIV protein genes or two or more herpes virus protein genes. Inother embodiments, they include guide strands having complementary totwo or more herpes simplex virus genes or mRNAs, e.g., the UL29 gene ormRNA and the Nectin-1 gene or mRNA of HSV-2, to reduce HSV-2 expression,replication or activity. In one embodiment, the polynucleotide complexesor molecules having regions targeting two or more HSV-2 genes or mRNAsare present in a formulation for topical delivery.

In particular embodiments, polynucleotide complexes and molecules of thepresent invention comprise one, two, three or more guide strands ortarget-specific regions that target an apolipoprotein B (ApoB) gene ormRNA, e.g., the human ApoB gene or mRNA or the mouse ApoB gene or mRNA.Accordingly, in particular embodiments, they comprise one, two, three ormore regions comprising a region complementary to a region of the humanApoB sequence set forth in SEQ ID NO:1. In other embodiments, theycomprise one, two, three or more regions comprising a regioncomplementary to a region of the mouse ApoB sequence set forth in SEQ IDNO:10. In particular embodiments, they comprise two or more guidesequences having the specific sequences set forth in the accompanyingExamples.

In certain embodiments, polynucleotide complexes and molecules of thepresent invention comprise one, two, three or more guide strands orregions that target HIV genes. In particular embodiments, they targetone, two, three or more HIV genes or mRNAs encoding one or more proteinsselected from HIV gag, HIV tat, HIV env, HIV gag-pol, HIV vif, and HIVnef proteins. Accordingly, in particular embodiments, they comprise one,two, three or more regions complementary to a region of the HIV gagsequence set forth in SEQ ID NO:2; one, two, three or more regionscomplementary to a region of the HIV tat sequence set forth in SEQ IDNO:3, one, two, three or more regions complementary to a region of theHIV env sequence set forth in SEQ ID NO:4, one, two, three or moreregions complementary to a region of the HIV gag-pol sequence set forthin SEQ ID NO:5, one, two, three or more regions comprising a regioncomplementary to a region of the HIV vif sequence set forth in SEQ IDNO:6, one, two, three or more regions comprising a region complementaryto a region of the HIV nef sequence set forth in SEQ ID NO:7. Inparticular embodiments, they comprise two or more guide sequences havingthe specific HIV sequences set forth in the accompanying Examples.

In certain embodiments, selection of a sequence region complementary toa target gene (or gene) is based upon analysis of the chosen targetsequence and determination of secondary structure, T_(m), bindingenergy, and relative stability and cell specificity. Such sequences maybe selected based upon their relative inability to form dimers,hairpins, or other secondary structures that would reduce structuralintegrity of the polynucleotide or prohibit specific binding to thetarget gene in a host cell.

Preferred target regions of the target gene or mRNA may include thoseregions at or near the AUG translation initiation codon and thosesequences that are substantially complementary to 5′ regions of the geneor mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402) orOligoengine Workstation 2.0.

In one embodiment, target sites are preferentially not located withinthe 5′ and 3′ untranslated regions (UTRs) or regions near the startcodon (within approximately 75 bases), since proteins that bindregulatory regions may interfere with the binding of the polynucleotideIn addition, potential target sites may be compared to an appropriategenome database, such as BLASTN 2.0.5, available on the NCBI server atwww.ncbi.nlm, and potential target sequences with significant homologyto other coding sequences eliminated.

In another embodiment, the target sites are located within the 5′ or 3′untranslated region (UTRs). In addition, the self-complementary regionof the polynucleotide may be composed of a particular sequence found inthe gene of the target.

The target gene may be of any species, including, for example, plant,animal (e.g. mammalian), protozoan, viral (e.g., HIV), bacterial orfungal. In certain embodiments, the polynucleotides of the presentinvention may comprise or be complementary to the GFP sequences inExample 1, the HIV sequences in Example 2, or the ApoB sequences inExample 3.

As noted above, the target gene sequence and the complementary region ofthe polynucleotide may be complete complements of each other, or theymay be less than completely complementary, as long as the strandshybridize to each other under physiological conditions.

The polynucleotide complexes and molecules of the present inventioncomprise at least one, two, or three regions complementary to one ormore target genes, as well as one or more self-complementary regionsand/or interconnecting loops, Typically, the region complementary to atarget gene is 15 to 17 to 24 nucleotides in length, including integervalues within these ranges. This region may be at least 16 nucleotidesin length, at least 17 nucleotides in length, at least 20 nucleotides inlength, at least 24 nucleotides in length, between 15 and 24 nucleotidesin length, between 16 and 24 nucleotides in length, or between 17 and 24nucleotides in length, inclusive of the end values, including anyinteger value within these ranges.

The self-complementary region is typically between 2 and 54 nucleotidesin length, at least 2 nucleotides in length, at least 16 nucleotides inlength, or at least 20 nucleotides in length, including any integervalue within any of these ranges. Hence, in one embodiment, aself-complementary region may comprise about 1-26 nucleotide pairs. Asingle-stranded region can be about 3-15 nucleotides, including allintegers in between. A null region refers to a region that isnot-specific for any target gene, at least by design. A null region orstrand may be used in place of a target-specific region, such as in thedesign of a bi-valent polynucleotide complex or molecule of theinvention (see, e.g., Figure IV(K)).

In certain embodiments, a self-complementary region is long enough toform a double-stranded structure. In certain embodiments, a 3′ regionand a 5′ region may hybridize to for a self-complementary region (i.e.,a double-stranded region) comprising a stem-loop structure. Accordingly,in one embodiment, the primary sequence of a self-complementary regioncomprises two stretches of sequence complementary to each otherseparated by additional sequence that is not complementary or issemi-complementary. While less optimal, the additional sequence can becomplementary in certain embodiments. The additional sequence forms theloop of the stem-loop structure and, therefore, must be long enough tofacilitate the folding necessary to allow the two complementarystretches to bind each other. In particular embodiments, the loopsequence comprises at least 3, at least 4, at least 5 or at least 6bases. In one embodiment, the loop sequence comprises 4 bases. The twostretches of sequence complementary to each other (within theself-complementary region; i.e., the stem regions) are of sufficientlength to specifically hybridize to each other under physiologicalconditions. In certain embodiments, each stretch comprises 4 to 12nucleotides; in other embodiments, each stretch comprises at least 4, atleast 5, at least 6, at least 8, or at least 10 nucleotides, or anyinteger value within these ranges. In a particular embodiment, aself-complementary region comprises two stretches of at least 4complementary nucleotides separated by a loop sequence of at least 4nucleotides. In certain embodiments, all or a portion of aself-complementary region may or may not be complementary to the regionof the polynucleotide that is complementary to the target gene or gene.

In particular embodiments, self-complementary regions possessthermodynamic parameters appropriate for binding of self-complementaryregions, e.g., to form a stem-loop structure.

In one embodiment, self-complementary regions are dynamically calculatedby use of RNA via free-energy analysis and then compared to the energycontained within the remaining “non self-complementary region” or loopregion to ensure that the energy composition is adequate to form adesired structure, e.g., a stem-loop structure. In general, differentnucleotide sequences of the gene targeting region are considered indetermining the compositions of the stem-loop structures to ensure theformation of such. The free-energy analysis formula may again be alteredto account for the type of nucleotide or pH of the environment in whichit is used. Many different secondary structure prediction programs areavailable in the art, and each may be used according to the invention.Thermodynamic parameters for RNA and DNA bases are also publiclyavailable in combination with target sequence selection algorithms, ofwhich several are available in the art.

In one embodiment, the polynucleotide complex or molecule comprises orconsists of (a) three oligonucleotides comprising 17 to 24 nucleotidesin length (including any integer value in-between), which iscomplementary to and capable of hybridizing under physiologicalconditions to at least a portion of an gene molecule, flanked optionallyby (b) self-complementary sequences comprising 16 to 54 nucleotides inlength (including any integer value in-between) or (c) 2 to 12nucleotides capable of forming a loop. In one embodiment, eachself-complementary sequence is capable of forming a stem-loop structure,one of which is located at the 5′ end and one of which is located at the3′ end of the secondary guide strands.

In certain embodiments, the self-complementary region functions as astructure to recruit enzymatic cleavage of itself and/or bind toparticular regions of proteins involved in the catalytic process of genemodulation, In addition, the loop may be of a certain 4-nucleotide(e.g., tetraloop NGNN, AAGU, UUGA, or GUUA) structure to promote thecleavage of the self-complementary region by an RNase such as RNase III.In addition, the self-complementary region can be cleaved by RNase III11/13 or 14/16 nucleotides into the duplex region leaving a 2 nucleotide3′ end. In certain embodiments, the tetraloop has the sequence GNRA orGNYA, where N indicates any nucleotide or nucleoside, R indicates apurine nucleotide or nucleoside; and Y indicates a pyrimidine nucleotideor nucleoside.

In certain embodiments, the self-complementary polynucleotide that hasbeen enzymatically cleaved as described above will load onto the proteinregion of RISC complexes. In certain embodiments, the self-complementaryregion containing a loop greater than 4 nucleotides can prevent thecleavage of the self-complementary region by RNase such as RNase III. Inpreferred embodiments, the polynucleotide of the present invention bindsto and reduces expression of a target gene. A target gene may be a knowngene target, or, alternatively, a target gene may be not known, i.e., arandom sequence may be used. In certain embodiments, target gene levelsare reduced at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, or at least 95%.

In one embodiment of the invention, the level of inhibition of targetgene expression (i.e., gene expression) is at least 90%, at least 95%,at least 98%, and at least 99% or is almost 100%, and hence the cell ororganism will in effect have the phenotype equivalent to a so-called“knock out” of a gene. However, in some embodiments, it may be preferredto achieve only partial inhibition so that the phenotype is equivalentto a so-called “knockdown” of the gene. This method of knocking downgene expression can be used therapeutically or for research (e.g., togenerate models of disease states, to examine the function of a gene, toassess whether an agent acts on a gene, to validate targets for drugdiscovery).

The polynucleotide complexes and molecules of the invention can be usedto target and reduce or inhibit expression of genes (inclusive of codingand non-coding sequences), cDNAs, mRNAs, or microRNAs. In particularembodiments, their guide strands or targeting regions bind to mRNAs ormicroRNAs.

The invention further provides arrays of the polynucleotide of theinvention, including microarrays. Microarrays are miniaturized devicestypically with dimensions in the micrometer to millimeter range forperforming chemical and biochemical reactions and are particularlysuited for embodiments of the invention. Arrays may be constructed viamicroelectronic and/or microfabrication using essentially any and alltechniques known and available in the semiconductor industry and/or inthe biochemistry industry, provided that such techniques are amenable toand compatible with the deposition and/or screening of polynucleotidesequences.

Microarrays of the invention are particularly desirable for highthroughput analysis of multiple polynucleotides. A microarray typicallyis constructed with discrete region or spots that comprise thepolynucleotide of the present invention, each spot comprising one ormore the polynucleotide, preferably at positionally addressablelocations on the array surface. Arrays of the invention may be preparedby any method available in the art. For example, the light-directedchemical synthesis process developed by Affymetrix (see, U.S. Pat. Nos.5,445,934 and 5,856,174) may be used to synthesize biomolecules on chipsurfaces by combining solid-phase photochemical synthesis withphotolithographic fabrication techniques. The chemical depositionapproach developed by Incyte Pharmaceutical uses pre-synthesized cDNAprobes for directed deposition onto chip surfaces (see, e.g., U.S. Pat.No. 5,874,554).

In certain embodiments, a polynucleotide molecule of the presentinvention is chemically synthesized using techniques widely available inthe art, and annealed as a three stranded complex. In a relatedembodiment, the three or more guide strands of a polynucleotide complexof the present invention may be individually chemically synthesized andannealed to produce the polynucleotide complex.

In other embodiments, it is expressed in vitro or in vivo usingappropriate and widely known techniques, such as vectors or plasmidconstructs. Accordingly, in certain embodiments, the present inventionincludes in vitro and in vivo expression vectors comprising the sequenceof a polynucleotide of the present invention interconnected by eitherstem-loop or loop forming nucleotide sequences. Methods well known tothose skilled in the art may be used to construct expression vectorscontaining sequences encoding a polynucleotide, as well as appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A vector or nucleic acid construct system can comprise a single vectoror plasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. In the present case, the vector or nucleic acidconstruct is preferably one which is operably functional in a mammaliancell. The vector can also include a selection marker such as anantibiotic or drug resistance gene, or a reporter gene (i.e., greenfluorescent protein, luciferase), that can be used for selection oridentification of suitable transformants or transfectants. Exemplarydelivery systems may include viral vector systems (i.e., viral-mediatedtransduction) including, but not limited to, retroviral (e.g.,lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors,and herpes viral vectors, among others known in the art.

As noted above, certain embodiments employ retroviral vectors such aslentiviral vectors. The term “lentivirus” refers to a genus of complexretroviruses that are capable of infecting both dividing andnon-dividing cells. Examples of lentiviruses include HIV (humanimmunodeficiency virus; including HIV type 1, and HIV type 2),visna-maedi, the caprine arthritis-encephalitis virus, equine infectiousanemia virus, feline immunodeficiency virus (FIV), bovine immunedeficiency virus (BIV), and simian immunodeficiency virus (SIV).Lentiviral vectors can be derived from any one or more of theselentiviruses (see, e.g., Evans et al., Hum Gene Ther. 10:1479-1489,1999; Case et al., PNAS USA 96:2988-2993, 1999; Uchida et al., PNAS USA95:11939-11944, 1998; Miyoshi et al., Science 283:682-686, 1999; Suttonet al., J Virol 72:5781-5788, 1998; and Frecha et al., Blood.112:4843-52, 2008, each of which is incorporated by reference in itsentirety).

In certain embodiments the retroviral vector comprises certain minimalsequences from a lentivirus genome, such as the HIV genome or the SIVgenome. The genome of a lentivirus is typically organized into a 5′ longterminal repeat (LTR) region, the gag gene, the pol gene, the env gene,the accessory genes (e.g., nef, vif, vpr, vpu, tat, rev) and a 3′ LTRregion. The viral LTR is divided into three regions referred to as U3, R(repeat) and U5. The U3 region contains the enhancer and promoterelements, the U5 region contains the polyadenylation signals, and the Rregion separates the U3 and U5 regions. The transcribed sequences of theR region appear at both the 5′ and 3′ ends of the viral RNA (see, e.g.,“RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., OxfordUniversity Press, 2000); O Narayan, J. Gen. Virology. 70:1617-1639,1989; Fields et al., Fundamental Virology Raven Press., 1990; Miyoshi etal., J Virol. 72:8150-7, 1998; and U.S. Pat. No. 6,013,516, each ofwhich is incorporated by reference in its entirety). Lentiviral vectorsmay comprise any one or more of these elements of the lentiviral genome,to regulate the activity of the vector as desired, or, they may containdeletions, insertions, substitutions, or mutations in one or more ofthese elements, such as to reduce the pathological effects of lentiviralreplication, or to limit the lentiviral vector to a single round ofinfection.

Typically, a minimal retroviral vector comprises certain 5′LTR and 3′LTRsequences, one or more genes of interest (to be expressed in the targetcell), one or more promoters, and a cis-acting sequence for packaging ofthe RNA. Other regulatory sequences can be included, as described hereinand known in the art. The viral vector is typically cloned into aplasmid that may be transfected into a packaging cell line, such as aeukaryotic cell (e.g., 293-HEK), and also typically comprises sequencesuseful for replication of the plasmid in bacteria.

In certain embodiments, the viral vector comprises sequences from the 5′and/or the 3′ LTRs of a retrovirus such as a lentivirus. The LTRsequences may be LTR sequences from any lentivirus from any species. Forexample, they may be LTR sequences from HIV, SIV, FIV or BIV. Preferablythe LTR sequences are HIV LTR sequences.

In certain embodiments, the viral vector comprises the R and U5sequences from the 5′ LTR of a lentivirus and an inactivated or“self-inactivating” 3′ LTR from a lentivirus. A “self-inactivating 3′LTR” is a 3′ long terminal repeat (LTR) that contains a mutation,substitution or deletion that prevents the LTR sequences from drivingexpression of a downstream gene. A copy of the U3 region from the 3′ LTRacts as a template for the generation of both LTR's in the integratedprovirus. Thus, when the 3′ LTR with an inactivating deletion ormutation integrates as the 5′ LTR of the provirus, no transcription fromthe 5′ LTR is possible. This eliminates competition between the viralenhancer/promoter and any internal enhancer/promoter. Self-inactivating3′ LTRs are described, for example, in Zufferey et al., J Virol.72:9873-9880, 1998; Miyoshi et al., J Virol. 72:8150-8157, 1998; andIwakuma et al., Virology 261:120-132, 1999, each of which isincorporated by reference in its entirety. Self-inactivating 3′ LTRs maybe generated by any method known in the art. In certain embodiments, theU3 element of the 3′ LTR contains a deletion of its enhancer sequence,preferably the TATA box, Spl and/or NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

Expression vectors typically include regulatory sequences, whichregulate expression of the polynucleotide. Regulatory sequences presentin an expression vector include those non-translated regions of thevector, e.g., enhancers, promoters, 5′ and 3′ untranslated regions,which interact with host cellular proteins to carry out transcriptionand translation. Such elements may vary in their strength andspecificity. Depending on the vector system and cell utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. In addition, tissue-or -cell specific promoters may also be used.

For expression in mammalian cells, promoters from mammalian genes orfrom mammalian viruses are generally preferred. In addition, a number ofviral-based expression systems are generally available. For example, incases where an adenovirus is used as an expression vector, sequencesencoding a polypeptide of interest may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Certain embodiments may employ the one or more of the RNA polymerase IIand III promoters. A suitable selection of RNA polymerase III promoterscan be found, for example, in Paule and White. Nucleic Acids Research.,Vol 28, pp 1283-1298, 2000, which is incorporated by reference in itsentirety. RNA polymerase II and III promoters also include any syntheticor engineered DNA fragments that can direct RNA polymerase II or III,respectively, to transcribe its downstream RNA coding sequences.Further, the RNA polymerase II or III (Pol II or III) promoter orpromoters used as part of the viral vector can be inducible. Anysuitable inducible Pol II or III promoter can be used with the methodsof the invention. Exemplary Pol II or III promoters include thetetracycline responsive promoters provided in Ohkawa and Taira, HumanGene Therapy, Vol. 11, pp 577-585, 2000; and Meissner et al., NucleicAcids Research, Vol. 29, pp 1672-1682, 2001, each of which isincorporated by reference in its entirety.

Non-limiting examples of constitutive promoters that may be used includethe promoter for ubiquitin, the CMV promoter (see, e.g., Karasuyama etal., J. Exp. Med. 169:13, 1989), the β-actin (see, e.g., Gunning et al.,PNAS USA 84:4831-4835, 1987), and the pgk promoter (see, e.g., Adra etal., Gene 60:65-74, 1987); Singer-Sam et al., Gene 32:409-417, 1984; andDobson et al., Nucleic Acids Res. 10:2635-2637, 1982, each of which isincorporated by reference). Non-limiting examples of tissue specificpromoters include the Ick promoter (see, e.g., Garvin et al., Mol. CellBiol. 8:3058-3064, 1988; and Takadera et al., Mol. Cell Biol.9:2173-2180, 1989), the myogenin promoter (Yee et al., Genes andDevelopment 7:1277-1289, 1993), and the thy1 (see, e.g., Gundersen etal., Gene 113:207-214, 1992).

Additional examples of promoters include the ubiquitin-C promoter, thehuman μ heavy chain promoter or the Ig heavy chain promoter (e.g.,MH-b12), and the human κ light chain promoter or the Ig light chainpromoter (e.g., EEK-b12), which are functional in B-lymphocytes. TheMH-b12 promoter contains the human μ heavy chain promoter preceded bythe iEμ enhancer flanked by matrix association regions, and the EEK-b12promoter contains the κ light chain promoter preceded an intronicenhancer (iEκ), a matrix associated region, and a 3′ enhancer (3′Eκ)(see, e.g., Luo et al., Blood. 113:1422-1431, 2009, herein incorporatedby reference). Accordingly, certain embodiments may employ one or moreof these promoter or enhancer elements.

In certain embodiments, the invention provides for the conditionalexpression of a polynucleotide. A variety of conditional expressionsystems are known and available in the art for use in both cells andanimals, and the invention contemplates the use of any such conditionalexpression system to regulate the expression or activity of apolynucleotide. In one embodiment of the invention, for example,inducible expression is achieved using the REV-TET system. Components ofthis system and methods of using the system to control the expression ofa gene are well documented in the literature, and vectors expressing thetetracycline-controlled transactivator (tTA) or the reverse tTA (rtTA)are commercially available (e.g., pTet-Off, pTet-On and ptTA-2/3/4vectors, Clontech, Palo Alto, Calif.). Such systems are described, forexample, in U.S. Pat. Nos. 5,650,298, 6,271,348, 5,922,927, and relatedpatents, which are incorporated by reference in their entirety.

In certain embodiments, the viral vectors (e.g., retroviral, lentiviral)provided herein are “pseudo-typed” with one or more selected viralglycoproteins or envelope proteins, mainly to target selected celltypes. Pseudo-typing refers to generally to the incorporation of one ormore heterologous viral glycoproteins onto the cell-surface virusparticle, often allowing the virus particle to infect a selected cellthat differs from its normal target cells. A “heterologous” element isderived from a virus other than the virus from which the RNA genome ofthe viral vector is derived. Typically, the glycoprotein-coding regionsof the viral vector have been genetically altered such as by deletion toprevent expression of its own glycoprotein. Merely by way ofillustration, the envelope glycoproteins gp41 and/or gp120 from anHIV-derived lentiviral vector are typically deleted prior topseudo-typing with a heterologous viral glycoprotein.

Generation of viral vectors can be accomplished using any suitablegenetic engineering techniques known in the art, including, withoutlimitation, the standard techniques of restriction endonucleasedigestion, ligation, transformation, plasmid purification, PCRamplification, and DNA sequencing, for example as described in Sambrooket al. (Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, N.Y. (1989)), Coffin et al. (Retroviruses. Cold SpringHarbor Laboratory Press, N.Y. (1997)) and “RNA Viruses: A PracticalApproach” (Alan J. Cann, Ed., Oxford University Press, (2000)).

Any variety of methods known in the art may be used to produce suitableretroviral particles whose genome comprises an RNA copy of the viralvector. As one method, the viral vector may be introduced into apackaging cell line that packages the viral genomic RNA based on theviral vector into viral particles with a desired target cellspecificity. The packaging cell line typically provides in trans theviral proteins that are required for packaging the viral genomic RNAinto viral particles and infecting the target cell, including thestructural gag proteins, the enzymatic pol proteins, and the envelopeglycoproteins.

In certain embodiments, the packaging cell line may stably expresscertain of the necessary or desired viral proteins (e.g., gag, pol)(see, e.g., U.S. Pat. No. 6,218,181, herein incorporated by reference).In certain embodiments, the packaging cell line may be transientlytransfected with plasmids that encode certain of the necessary ordesired viral proteins (e.g., gag, pol, glycoprotein), including themeasles virus glycoprotein sequences described herein. In one exemplaryembodiment, the packaging cell line stably expresses the gag and polsequences, and the cell line is then transfected with a plasmid encodingthe viral vector and a plasmid encoding the glycoprotein. Followingintroduction of the desired plasmids, viral particles are collected andprocessed accordingly, such as by ultracentrifugation to achieve aconcentrated stock of viral particles. Exemplary packaging cell linesinclude 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK(ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cell lines.

In one particular embodiment, the polynucleotides are expressed using avector system comprising a pSUPER vector backbone and additionalsequences corresponding to the polynucleotide to be expressed. ThepSUPER vectors system has been shown useful in expressing shRNA reagentsand downregulating gene expression (Brummelkamp, T. T. et al., Science296:550 (2002) and Brummelkamp, T. R. et al., Cancer Cell, publishedonline Aug. 22, 2002). PSUPER vectors are commercially available fromOligoEngine, Seattle, Wash.

Methods of Regulating Gene Expression

The polynucleotides of the invention may be used for a variety ofpurposes, all generally related to their ability to inhibit or reduceexpression of one or more target genes. Accordingly, the inventionprovides methods of reducing expression of one or more target genescomprising introducing a polynucleotide complex or molecule of thepresent invention into a cell comprising said one or more target genes.In particular embodiments, the polynucleotide complex or moleculecomprises one or more guide strands that collectively target the one ormore target genes. In one embodiment, a polynucleotide of the inventionis introduced into a cell that contains a target gene or a homolog,variant or ortholog thereof, targeted by either one, two, or three ofthe guide strands or targeting regions.

In addition, the polynucleotides of the present invention may be used toreduce expression indirectly. For example, a polynucleotide complex ormolecule of the present invention may be used to reduce expression of atransactivator that drives expression of a second gene (i.e., the targetgene), thereby reducing expression of the second gene. Similarly, apolynucleotide may be used to increase expression indirectly. Forexample, a polynucleotide complex or molecule of the present inventionmay be used to reduce expression of a transcriptional repressor thatinhibits expression of a second gene, thereby increasing expression ofthe second gene.

In various embodiments, a target gene is a gene derived from the cellinto which a polynucleotide is to be introduced, an endogenous gene, anexogenous gene, a transgene, or a gene of a pathogen that is present inthe cell after transfection thereof. Depending on the particular targetgene and the amount of the polynucleotide delivered into the cell, themethod of this invention may cause partial or complete inhibition of theexpression of the target gene. The cell containing the target gene maybe derived from or contained in any organism (e.g., plant, animal,protozoan, virus, bacterium, or fungus). As used herein, “target genes”include genes, mRNAs, and microRNAs.

Inhibition of the expression of the target gene can be verified by meansincluding, but not limited to, observing or detecting an absence orobservable decrease in the level of protein encoded by a target gene, anabsence or observable decrease in the level of a gene product expressedfrom a target gene (e.g., mRNA0, and/or a phenotype associated withexpression of the gene, using techniques known to a person skilled inthe field of the present invention.

Examples of cell characteristics that may be examined to determine theeffect caused by introduction of a polynucleotide complex or molecule ofthe present invention include, cell growth, apoptosis, cell cyclecharacteristics, cellular differentiation, and morphology.

A polynucleotide complex or molecule of the present invention may bedirectly introduced to the cell (i.e., intracellularly), or introducedextracellularly into a cavity or interstitial space of an organism,e.g., a mammal, into the circulation of an organism, introduced orally,introduced by bathing an organism in a solution containing thepolynucleotide, or by some other means sufficient to deliver thepolynucleotide into the cell.

In addition, a vector engineered to express a polynucleotide may beintroduced into a cell, wherein the vector expresses the polynucleotide,thereby introducing it into the cell. Methods of transferring anexpression vector into a cell are widely known and available in the art,including, e.g., transfection, lipofection, scrape-loading,electroporation, microinjection, infection, gene gun, andretrotransposition. Generally, a suitable method of introducing a vectorinto a cell is readily determined by one of skill in the art based uponthe type of vector and the type of cell, and teachings widely availablein the art. Infective agents may be introduced by a variety of meansreadily available in the art, including, e.g., nasal inhalation.

Methods of inhibiting gene expression using the oligonucleotides of theinvention may be combined with other knockdown and knockout methods,e.g., gene targeting, antisense RNA, ribozymes, double-stranded RNA(e.g., shRNA and siRNA) to further reduce expression of a target gene.

In different embodiments, target cells of the invention are primarycells, cell lines, immortalized cells, or transformed cells. A targetcell may be a somatic cell or a germ cell. The target cell may be anon-dividing cell, such as a neuron, or it may be capable ofproliferating in vitro in suitable cell culture conditions. Target cellsmay be normal cells, or they may be diseased cells, including thosecontaining a known genetic mutation. Eukaryotic target cells of theinvention include mammalian cells, such as, for example, a human cell, amurine cell, a rodent cell, and a primate cell. In one embodiment, atarget cell of the invention is a stem cell, which includes, forexample, an embryonic stem cell, such as a murine embryonic stem cell.

The polynucleotide complexes, molecules, and methods of the presentinvention may be used to treat any of a wide variety of diseases ordisorders, including, but not limited to, inflammatory diseases,cardiovascular diseases, nervous system diseases, tumors, demyelinatingdiseases, digestive system diseases, endocrine system diseases,reproductive system diseases, hemic and lymphatic diseases,immunological diseases, mental disorders, muscoloskeletal diseases,neurological diseases, neuromuscular diseases, metabolic diseases,sexually transmitted diseases, skin and connective tissue diseases,urological diseases, and infections.

In certain embodiments, the methods are practiced on an animal, inparticular embodiments, a mammal, and in certain embodiments, a human.

Accordingly, in one embodiment, the present invention includes methodsof using a polynucleotide complex or molecule of the present inventionfor the treatment or prevention of a disease associated with genederegulation, overexpression, or mutation. For example, a polynucleotidecomplex or molecule of the present invention may be introduced into acancerous cell or tumor and thereby inhibit expression of a generequired for or associated with maintenance of thecarcinogenic/tumorigenic phenotype. To prevent a disease or otherpathology, a target gene may be selected that is, e.g., required forinitiation or maintenance of a disease/pathology. Treatment may includeamelioration of any symptom associated with the disease or clinicalindication associated with the pathology.

In addition, the polynucleotides of the present invention are used totreat diseases or disorders associated with gene mutation. In oneembodiment, a polynucleotide is used to modulate expression of a mutatedgene or allele. In such embodiments, the mutated gene is a target of thepolynucleotide complex or molecule, which will comprise a regioncomplementary to a region of the mutated gene. This region may includethe mutation, but it is not required, as another region of the gene mayalso be targeted, resulting in decreased expression of the mutant geneor gene. In certain embodiments, this region comprises the mutation,and, in related embodiments, the polynucleotide complex or moleculespecifically inhibits expression of the mutant gene or gene but not thewild type gene or gene. Such a polynucleotide is particularly useful insituations, e.g., where one allele is mutated but another is not.However, in other embodiments, this sequence would not necessarilycomprise the mutation and may, therefore, comprise only wild-typesequence. Such a polynucleotide is particularly useful in situations,e.g., where all alleles are mutated. A variety of diseases and disordersare known in the art to be associated with or caused by gene mutation,and the invention encompasses the treatment of any such disease ordisorder with a the polynucleotide.

In certain embodiments, a gene of a pathogen is targeted for inhibition.For example, the gene could cause immunosuppression of the host directlyor be essential for replication of the pathogen, transmission of thepathogen, or maintenance of the infection. In addition, the target genemay be a pathogen gene or host gene responsible for entry of a pathogeninto its host, drug metabolism by the pathogen or host, replication orintegration of the pathogen's genome, establishment or spread of aninfection in the host, or assembly of the next generation of pathogen.Methods of prophylaxis (i.e., prevention or decreased risk ofinfection), as well as reduction in the frequency or severity ofsymptoms associated with infection, are included in the presentinvention. For example, cells at risk for infection by a pathogen oralready infected cells, particularly human immunodeficiency virus (HIV)infections, may be targeted for treatment by introduction of a thepolynucleotide according to the invention (see Examples 1 and 2 fortargeting sequences). Thus, in one embodiment, polynucleotide complexesor molecules of the present invention that target one or more HIVproteins are used to treat or inhibit HIV infection or acquired immunedeficiency syndrome (AIDS).

In other specific embodiments, the present invention is used for thetreatment or development of treatments for cancers of any type. Examplesof tumors that can be treated using the methods described hereininclude, but are not limited to, neuroblastomas, myelomas, prostatecancers, small cell lung cancer, colon cancer, ovarian cancer, non-smallcell lung cancer, brain tumors, breast cancer, leukemias, lymphomas, andothers.

In one embodiment, polynucleotide complexes or molecules of the presentinvention that target apolipoprotein B (apoB) are used to treat, reduce,or inhibit atherosclerosis or heart disease. ApoB is the primaryapolipoprotein of low-density lipoproteins (LDLs), which is responsiblefor carrying cholesterol to tissues. ApoB on the LDL particle acts as aligand for LDL receptors, and high levels of ApoB can lead to plaquesthat cause vascular disease (atherosclerosis), leading to heart disease.

The polynucleotide complexes, molecules and expression vectors(including viral vectors and viruses) may be introduced into cells invitro or ex vivo and then subsequently placed into an animal to affecttherapy, or they may be directly introduced to a patient by in vivoadministration. Thus, the invention provides methods of gene therapy, incertain embodiments. Compositions of the invention may be administeredto a patient in any of a number of ways, including parenteral,intravenous, systemic, local, topical, oral, intratumoral,intramuscular, subcutaneous, intraperitoneal, inhalation, or any suchmethod of delivery. In one embodiment, the compositions are administeredparenterally, i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In a specific embodiment, theliposomal compositions are administered by intravenous infusion orintraperitoneally by a bolus injection.

Compositions of the invention may be formulated as pharmaceuticalcompositions suitable for delivery to a subject. The pharmaceuticalcompositions of the invention will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose, dextrose or dextrans),mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The amount of the oligonucleotides administered to a patient can bereadily determined by a physician based upon a variety of factors,including, e.g., the disease and the level of the oligonucleotidesexpressed from the vector being used (in cases where a vector isadministered). The amount administered per dose is typically selected tobe above the minimal therapeutic dose but below a toxic dose. The choiceof amount per dose will depend on a number of factors, such as themedical history of the patient, the use of other therapies, and thenature of the disease. In addition, the amount administered may beadjusted throughout treatment, depending on the patient's response totreatment and the presence or severity of any treatment-associated sideeffects.

Methods of Determining Gene Function

The invention further includes a method of identifying gene function inan organism comprising the use of a polynucleotide complex or moleculeof the present invention to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics envisions determining the function of uncharacterized genes byemploying the invention to reduce the amount and/or alter the timing oftarget gene activity. The invention may be used in determining potentialtargets for pharmaceutics, understanding normal and pathological eventsassociated with development, determining signaling pathways responsiblefor postnatal development/aging, and the like. The increasing speed ofacquiring nucleotide sequence information from genomic and expressedgene sources, including total sequences for the yeast, D. melanogaster,and C. elegans genomes, can be coupled with the invention to determinegene function in an organism (e.g., nematode). The preference ofdifferent organisms to use particular codons, searching sequencedatabases for related gene products, correlating the linkage map ofgenetic traits with the physical map from which the nucleotide sequencesare derived, and artificial intelligence methods may be used to defineputative open reading frames from the nucleotide sequences acquired insuch sequencing projects.

In one embodiment, a polynucleotide of the present invention is used toinhibit gene expression based upon a partial sequence available from anexpressed sequence tag (EST), e.g., in order to determine the gene'sfunction or biological activity. Functional alterations in growth,development, metabolism, disease resistance, or other biologicalprocesses would be indicative of the normal role of the ESTs geneproduct.

The ease with which a polynucleotide can be introduced into an intactcell/organism containing the target gene allows the present invention tobe used in high throughput screening (HTS). For example, solutionscontaining the polynucleotide that are capable of inhibiting differentexpressed genes can be placed into individual wells positioned on amicrotiter plate as an ordered array, and intact cells/organisms in eachwell can be assayed for any changes or modifications in behavior ordevelopment due to inhibition of target gene activity. The function ofthe target gene can be assayed from the effects it has on thecell/organism when gene activity is inhibited. In one embodiment, thepolynucleotides of the invention are used for chemocogenomic screening,i.e., testing compounds for their ability to reverse a disease modeledby the reduction of gene expression using a polynucleotide of theinvention.

If a characteristic of an organism is determined to be geneticallylinked to a polymorphism through RFLP or QTL analysis, the presentinvention can be used to gain insight regarding whether that geneticpolymorphism might be directly responsible for the characteristic. Forexample, a fragment defining the genetic polymorphism or sequences inthe vicinity of such a genetic polymorphism can be amplified to producean RNA, a polynucleotide can be introduced to the organism, and whetheran alteration in the characteristic is correlated with inhibition can bedetermined.

The present invention is also useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of a thepolynucleotide at specific times of development and locations in theorganism without introducing permanent mutations into the target genome.Similarly, the invention contemplates the use of inducible orconditional vectors that express a the polynucleotide only when desired.

The present invention also relates to a method of validating whether agene product is a target for drug discovery or development. A thepolynucleotide that targets the gene that corresponds to the gene fordegradation is introduced into a cell or organism. The cell or organismis maintained under conditions in which degradation of the gene occurs,resulting in decreased expression of the gene. Whether decreasedexpression of the gene has an effect on the cell or organism isdetermined. If decreased expression of the gene has an effect, then thegene product is a target for drug discovery or development.

Methods of Designing and Producing Polynucleotide Complexes andMolecules

The polynucleotide complexes and molecules of the present inventioncomprise a novel and unique set of functional sequences, arranged in amanner so as to adopt a secondary structure containing one or moredouble-stranded regions (sometimes adjoined by stem-loop or loopstructures), which imparts the advantages of the polynucleotide.Accordingly, in certain embodiments, the present invention includesmethods of designing the polynucleotide complexes and molecules of thepresent invention. Such methods typically involve appropriate selectionof the various sequence components of the polynucleotide complexes andmolecules. The terms “primary strand”, “secondary strand”, and “keystrand” refer to the various guide strands present within apolynucleotide complex or molecule of the present invention.

In one embodiment, the basic design of the polynucleotide complex is asfollows:

Design Motifs:

(primary strand)(UU)(secondary strand)(UU)(key strand)(UU)

Accordingly, in a related embodiment, a the polynucleotide is designedas follows:

II. (secondary strand)(UU)(UU)(key strand)(UU)(primary strand)

III. (secondary strand)(UU)(loop or stem-loop)(key strand)(UU)(loop orstem-loop)(primary strand)(UU)

Set Parameters

Set seed size for self-complementarity at approximately 38-43%. For a 19nucleotide targets, a range or 7 or 8 nucleotides is preferred asSEED_SIZE.

For each gene, define a PRIMARY and SECONDARY target gene.

Define Primary Strands

Start with one or more target gene sequences. For each gene, build alist of PRIMARY target sequences 17-24 nucleotide motifs that meetcriteria of G/C content, specificity, and poly-A or poly-G free. Foreach, find also a SECONDARY and KEY strand.

Find Secondary and Key Strands

d. For each target sequence on each gene, clustal align base 1 throughSEED_SIZE the reverse of each sequence to the SECONDARY gene

Record sequence with a perfect alignment. The target sequence on theSECONDARY gene is the alignment start, minus the length of the motif,plus SEED_SIZE to alignment start, plus SEED_SIZE. The SECONDARY strandis the reverse compliment.

To find each KEY strand, define SEED_A as base 1 through SEED_SIZE ofthe PRIMARY strand, define SEED_B as bases at motif length minusSEED_SIZE to motif length of the SECONDARY strand. Set a MID_SECTION ascharacters “I” repeated of length motif sequence length minus SEED_Alength plus SEED_B length. Set key alignment sequence as SEED_A,MID_SECTION, SEED_B. Clustal align to the target gene for the keysegment. Record KEY target sequence as bases at alignment hit on keytarget gene to bases alignment hit plus motif length. The KEY strand isthe reverse compliment.

Construct Optional Polynucleotide

g. Build candidate Stem A & B with (4-24) nucleotides that have meltingtemperature dominant to equal length region of target. Stem strands haveA-T, G-C complementarity to each other. Length and composition dependupon which endoribonuclease is chosen for pre-processing of thestem-loop structure.

h. Build candidate Stem C & D with (4-24) nucleotides that have meltingtemperature dominant to equal length region of target. Stem strands haveA-T, G-C complementarity to each other, but no complementarity to Stem A& B. Length and composition depend upon which endoribonuclease is chosenfor pre-processing of the stem-loop structure.

i. Build loop candidates with (4-12) A-T rich nucleotides into loop A &B. Length and composition depend upon which endoribonuclease is chosenfor pre-processing of the stem-loop structure. Tetraloops as describedare suggested for longer stems processed by RNase III or Pac1 RNase IIIendoribonucleases as drawn in (Fig. A.). Larger loops are suggested forpreventing RNase III or Pac1 processing and placed onto shorter stems asdrawn in (Fig. C, Fig. D.).

j. Form a contiguous sequence for each motif candidate.

k. Fold candidate sequence using software with desired parameters.

l. From output, locate structures with single stranded target regionswhich are flanked at either one or both ends with a desired stem/loopstructure.

In one embodiment, a method of designing a polynucleotide sequencecomprising one or more self-complementary regions for the regulation ofexpression of a target gene (i.e., a the polynucleotide), includes: (a)selecting a first sequence 17 to 30 nucleotides in length andcomplementary to a target gene; and (b) selecting one or more additionalsequences 12 to 54 nucleotides in length, which comprisesself-complementary regions and which are non-complementary to the firstsequence.

These methods, in certain embodiments, include determining or predictingthe secondary structure adopted by the sequences selected in step (b),e.g., in order to determine that they are capable of adopting astem-loop structure.

Similarly, these methods can include a verification step, whichcomprises testing the designed polynucleotide sequence for its abilityto inhibit expression of a target gene, e.g., in an in vivo or in vitrotest system.

The invention further contemplates the use of a computer program toselect sequences of a polynucleotide, based upon the complementaritycharacteristics described herein. The invention, thus, provides computersoftware programs, and computer readable media comprising said softwareprograms, to be used to select the polynucleotide sequences, as well ascomputers containing one of the programs of the present invention.

In certain embodiments, a user provides a computer with informationregarding the sequence, location or name of a target gene. The computeruses this input in a program of the present invention to identify one ormore appropriate regions of the target gene to target, and outputs orprovides complementary sequences to use in the polynucleotide of theinvention. The computer program then uses this sequence information toselect sequences of the one or more self-complementary regions of thepolynucleotide. Typically, the program will select a sequence that isnot complementary to a genomic sequence, including the target gene, orthe region of the polynucleotide that is complementary to the targetgene. Furthermore, the program will select sequences ofself-complementary regions that are not complementary to each other.When desired, the program also provides sequences of gap regions. Uponselection of appropriate sequences, the computer program outputs orprovides this information to the user.

The programs of the present invention may further use input regardingthe genomic sequence of the organism containing the target gene, e.g.,public or private databases, as well as additional programs that predictsecondary structure and/or hybridization characteristics of particularsequences, in order to ensure that the polynucleotide adopts the correctsecondary structure and does not hybridize to non-target genes.

The present invention is based, in part, upon the surprising discoverythat the polynucleotide, as described herein, is extremely effective inreducing target gene expression of one or more genes. The polynucleotideoffer significant advantages over previously described antisense RNAs,including increased potency, and increased effectiveness to multipletarget genes. Furthermore, the polynucleotide of the invention offeradditional advantages over traditional dsRNA molecules used for siRNA,since the use of the polynucleotide substantially eliminates theoff-target suppression associated with dsRNA molecules and offersmultivalent RNAi.

It is understood that the compositions and methods of the presentinvention may be used to target a variety of different target genes. Theterm “target gene” may refer to a gene, an mRNA, or a microRNA.Accordingly, target sequences provided herein may be depicted as eitherDNA sequences or RNA sequences. One of skill the art will appreciatethat the compositions of the present invention may include regionscomplementary to either the DNA or RNA sequences provided herein. Thus,where either a DNA or RNA target sequence is provided, it is understoodthat the corresponding RNA or DNA target sequence, respectively, mayalso be targeted.

The practice of the present invention will employ a variety ofconventional techniques of cell biology, molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are fully described in the literature. See, forexample, Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., ed. bySambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press,1989); and DNA Cloning, Volumes I and II (D. N. Glover ed. 1985).

All of the patents, patent applications, and non-patent referencesreferred to herein are incorporated by reference in their entirety, asif each one was individually incorporated by reference.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

EXAMPLES Example 1 Trivoid Anti-GFP

Multivalent siRNA were designed against a single gene, the greenfluorescent protein (GFP). A multivalent synthetic RNA MV-siRNA complexdirected against GFP was tested to compare suppression activity inrelation to that of a single shRNA clone. Also, to test the effect ofdeactivating one of the strands of the synthetic MV-siRNA complex, onestrand was replaced with DNA (T1-19_C_dna); as shown below. Thisreplacement resulted in a relative drop in suppression by ˜30%.Additionally, ‘short’ and ‘long’ forms of the MV-siRNAself-complementary clones described herein were tested and compared tothe suppression of GFP expression in relation to that of a publishedshRNA clone.

Oligomer sequences for the synthetic MV-siRNA, and the DNA replacementstrand, are shown below in Table 1. The targeted regions of the GFPcoding sequence are illustrated in FIG. 8A.

TABLE 1 Oligos for Synthetic MV-siRNA: Name Sequence SEQ ID NO:TI-19/7_A GGGCAGCUUGCCGGUGGUGUU 11 TI-19/7_B CACCACCCCGGUGAACAGCUU 12TI-19/70 GCUGUUCACGUCGCUGCCCUU 13 TI-19/7_C_dna GCTGTTCACGTCGCTGCCC 14

To prepare the synthetic multivalent-siRNAs (MV-siRNAs), each tube ofthe individual oligos above was resuspended in RNase-free water toobtain a final concentration of 50 μM (50 pmoles/μL). The individualoligos were then combined as (a) TI-19/7_A, TI-19/7_B, and TI-19/7_C(MV-siRNA GFP I), or as (b) TI-19/7_A, TI-19/7_B, and TI-19/7_C_dna(MV-siRNA GFP I DNA), and annealed as follows. 30 μL of each one of theresupended oligos were combined with 10 μL of 10× annealing buffer (100mM Tris-HCl pH7.5, 1M NaCl, 10 mM EDTA), vortexed, heated for 5 minutesat 94° C., and step cooled to 70° C. over 30 minutes. The finalconcentration of the annealed MV-siRNA was about 15 μM.

To prepare the multivalent-siRNA clones and shRNA control, the sequencesin Table 2 below were cloned into the pSUPER vector, according to thepSUPER manual. The first sequence for each named clone (e.g., TI,T1_long, TII) represents the sequence of the self-complementarymultivalent siRNA that was expressed in the cell as an RNA transcript(comparable to the sequence of the synthetic MV-siRNAs in Table 1), andthe sequence referred to as “_as” is part of the coding sequence forthat molecule.

TABLE 2 Oligos for MV-siRNA expressing clones: Name Sequence SEQ ID NO:T1 GATCCCCCACCACCCCGGTGAACAGCgttaGCTGTTCACGTCGCT 15GCCCgttaGGGCAGCTTGCCGGTGGTGttTTTTTA TI_asAGCTTAACACCACCGGCAAGCTGCCCTAACGGGCAGCGACGTGAA 16CAGCTAACGCTGTTCACCGGGGTGGTGGGG T1_longGATCCCCCACCACCCCGGTGAACAGCTTGTAGGTGGCATCGCAGA 17AGCGATGCCACCTACAAGCTGTTCACGTCGCTGCCCTTGTAGGTGGCATCGCAGAAGCGATGCCACCTACAAGGGCAGCTTGCCGGTGGT GttTTTTTA T1_long_asAGCTTAACACCACCGGCAAGCTGCCCTTGTAGGTGGCATCGCTTC 18TGCGATGCCACCTACAAGGGCAGCGACGTGAACAGCTTGTAGGTGGCATCGCTTCTGCGATGCCACCTACAAGCTGTTCACCGGGGTGGT GGGG TIIGATCCCCCGTGCTGCTTCATGTGGTCGTTgttaCGACCACAATGG 19CGACAACCTTgttaGGTTGTCGGGCAGCAGCACGTTttTTTTTA TII_asAGCTTAAAACGTGCTGCTGCCCGACAACCTAACAAGGTTGTCGCC 20ATTGTGGTCGTAACAACGACCACATGAAGCAGCACGGGG TII_longGATCCCCCGTGCTGCTTCATGTGGTCGTTGTAGGTGGCATCGCAG 21AAGCGATGCCACCTACAACGACCACAATGGCGACAACCTTGTAGGTGGCATCGCAGAAGCGATGCCACCTACAAGGTTGTCGGGCAGCAG CACGttTTTTTA TII_long_asAGCTTAACGTGCTGCTGCCCGACAACCTTGTAGGTGGCATCGCTT 22CTGCGATGCCACCTACAAGGTTGTCGCCATTGTGGTCGTTGTAGGTGGCATCGCTTCTGCGATGCCACCTACAACGACCACATGAAGCAG CACGGGG shRNAGATCCCCGCAAGCTGACCCTGAAGTTCTTCAAGAGAGAACTTCAG 23 GGTCAGCTTGCTTTTTAshRNA_as AGCTTAAAAAGCAAGCTGACCCTGAAGTTCTOTCTTGAAGAACTT 24CAGGGTCAGCTTGCGGG

To test the effects on GFP-expression, the annealed MV-siRNA molecules(at a final concentration of 7.5 nM per well) and pSUPER vectorscontaining the MV-siRNA clones or shRNA control were transfected withLipofectamine 2000 into 293 cells that constitutively express GFP. GFPfluorescence was measure by flow cytometry 24 hour after transfection.

The results for one experiment are shown in Table 3 below, andsummarized in FIG. 7A. In FIG. 7A, the MV-siRNA long I and long IIclones demonstrate significantly increased suppression of GFP activitycompared to the shRNA control (referred to in that Figure as “siRNA”).

TABLE 3 Well Transfected: Mean Fluorescence % GFP Positive shRNA shRNA330 66% shRNA 302 60% Synthetic: MV-siRNA 305 61% Clone: MV-siRNA shortTI 360 72% MV-siRNA long TI 218 43% MV-siRNA long TII 245 49% NegativeBlank 502 100% non-GFP 293 cells 0.5 0%

FIG. 7B shows the results of an experiment in which the syntheticMV-siRNA GFP I complex demonstrated increased suppression of GFPactivity compared to the shRNA clone (referred to in that Figure as“siRNA”). However, the suppression activity for the MV-siRNA GFP Icomplex was slightly reduced when one strand was replaced with DNA, asshown for the synthetic MV-siRNA GFP I DNA complex.

Exemplary synthetic MV-siRNAs directed to GFP can also be designed as inTable 4 below, in which the 3 oligos of T1.A-C can be annealed asdescribed above. Similarly, the 3 oligos of T2.A-C can be annealed asdescribed above.

TABLE 4 Exemplary synhetic siRNA sets T1 and T2. Name SequenceSEQ ID NO: T1.A CUGCUGGUAGUGGUCGGCGUU 25 T1.B CGCCGACUUCGUGACGUGCUU 26T1.C GCACGUCGCCGUCCAGCAGUU 27 T2.A GUUGCCGUCGUCCUUGAAGUU 28 T2.BCUUCAAGUGGAACUACGGCUU 29 T2.C GCCGUAGGUAGGCGGCAACUU 30

MV-siRNA clones directed to GFP can also be designed as in Table 5below. As illustrated above, these sequences can be cloned into thepSuper vector, or any other vector system.

TABLE 5 Exemplary MV-siRNA clones Name Sequence SEQ ID NO: T1_transcriptCGCCGACUUCGUGACGUGCUUGUGCACGUCGCCGUCCAGCAG 31 UUGUCUGCUGGUAGUGGUCGGCGUUT1 GATCCCCCGCCGACTTCGTGACGTGCTTGTGCACGTCGCCGT 32CCAGCAGTTGTCTGCTGGTAGTGGTCGGCGTTTTTTTA T1_asAGCTTAAAAAAACGCCGACCACTACCAGCAGACAACTGCTGG 33ACGGCGACGTGCACAAGCACGTCACGAAGTCGGCGGGG T1_longCGCCGACUUCGUGACGUGCUUGUAGGUGGCAUCGCAGAAGCG 34 transcriptAUGCCACCUACAAGCACGUCGCCGUCCAGCAGUUGUAGGUGGCAUCGCAGAAGCGAUGCCACCUACAACUGCUGGUAGUGGUCG GCGUU T1_longGATCCCCCGCCGACTTCGTGACGTGCTTGTAGGTGGCATCGC 35AGAAGCGATGCCACCTACAAGCACGTCGCCGTCCAGCAGTTGTAGGTGGCATCGCAGAAGCGATGCCACCTACAACTGCTGGTA GTGGTCGGCGTTTTTA T1_long_asAGCTTAAAAACGCCGACCACTACCAGCAGTTGTAGGTGGCAT 36CGCTTCTGCGATGCCACCTACAACTGCTGGACGGCGACGTGCTTGTAGGTGGCATCGCTTCTGCGATGCCACCTACAAGCACGT CACGAAGTCGGCGGGGT2_transcript CUUCAAGUGGAACUACGGCUUGUGCCGUAGGUAGGCGGCAAC 37UUGUGUUGCCGUCGUCCUUGAAGUU T2 GATCCCCGGATCCGACATCCACGTTCTTCAAGAGAGAACGTG38 GATGTCGGATCCTTTTTA T2_as AGCTTAAAAAGGATCCGACATCCACGTTCTCTCTTGAAGAAC39 GTGGATGTCGGATCCGGG T2_long CUUCAAGUGGAACUACGGCUUGUAGGUGGCAUCGCAGAAGCG40 transcript AUGCCACCUACAAGCCGUAGGUAGGCGGCAACUUGUAGGUGGCAUCGCAGAAGCGAUGCCACCUACAAGUUGCCGUCGUCCUUG AAGUU T2_longGATCCCCCTTCAAGTGGAACTACGGCTTGTAGGTGGCATCGC 41AGAAGCGATGCCACCTACAAGCCGTAGGTAGGCGGCAACTTGTAGGTGGCATCGCAGAAGCGATGCCACCTACAAGTTGCCGTC GTCCTTGAAGTTTTTA T2_long_asAGCTTAAAAACTTCAAGGACGACGGCAACTTGTAGGTGGCAT 42CGCTTCTGCGATGCCACCTACAAGTTGCCGCCTACCTACGGCTTGTAGGTGGCATCGCTTCTGCGATGCCACCTACAAGCCGTA GTTCCACTTGAAGGGG

Example 2 Trivoid Anti-HIV

Multivalent-siRNA can be designed against multiple genes at unrelatedsites. In this example, a cloned MV-siRNA was tested against HIV. Theseresults show that a di-valent MV-siRNA molecule against HIV's Gag andTat (hv_sB) genes was significantly more efficient in inhibiting HIVreplication than an siRNA directed against Gag alone (hv_s).

The oligos shown in Table 6 were cloned into pSUPER.neo+gfp vectoraccording to manufacturer's guidelines. The hv_s is targeted to Gagonly, and the hv_sB is targeted to both Gag and Tat.

TABLE 6 Anti-HIV MV-siRNA clones Name Sequence SEQ ID NO: hv_sGATCCCCGTGAAGGGGAACCAAGAGATTgaTCTCTTGTTAATATCAG 43CTTgaGCTGATATTTCTCCTTCACTTTTTA hv_s_asAGCTTAAAAAGTGAAGGAGAAATATCAGCTCAAGCTGATATTAACAA 44GAGATCAATCTCTTGGTTCCCCTTCACGGG hv_sBGATCCCCCAAGCAGTTTTAGGCTGACgTTaGTCAGCCTCATTGACAC 45AGgTTaCTGTGTCAGCTGCTGOTTGTTTTTTTA hv_sB_AsAGCTTAAAAAAACAAGCAGCAGCTGACACAGTAACCTGTGTCAATGA 46GCCTGACTAACGTCAGCCTAAAACTGCTTGGGG

The vector constructs encoding the MV-siRNA clones were transfected intocells, and the analyses were carried out on days 10 and 40 postinfection with HIV-1 (pNL4.3 strain) with an MOI of 1.0. FIG. 9 showsthat at 10 days post transfection, inhibition of HIV replication by theMV-siRNA targeted to both Gag and Tat was about 3 times greater thaninhibition by the siRNA molecule targeted only to Gag.

Multivalent-siRNA can be designed to target 1, 2, or 3 different genesof HIV. The sequence of an exemplary HIV genome is provided in FIGS.10A-D. A sequence of an env gene is provided in FIG. 11, a gag gene inFIG. 12A, and a tat gene in FIG. 12B. The various genes or regions ofHIV can be generally defined and targeted by their range of nucleotidesequence as follows: 5′ LTR: 1-181; GAG: 336-1838; POL: 1631-4642; VIF:4587/4662-5165; VPR: 5105-5395 (including mutations at 5157, 5266, and5297); TAT: 5376-7966; REV: 5515-8195; VPU: 5607-5852; ENV: 5767-8337;NEF: 8339-8959; and 3′ LTR: 8628-9263. Based on these target genes,exemplary MV-RNA oligo sequences for HIV are provided in Table 7 below.

TABLE 7 Exemplary Trivalent MV-siRNA Sequences No. Sequence Target GeneSEQ ID NO:  1 GCCUUCCCUUGUGGGAAGGUU 1649  47  2 CCUUCCCUUGUGGGAAGGCUU1648  48  3 GCCUUCCUUGUGGGAAGGCUU 1648  49  4 UUCUGCACCUUACCUCUUAUU 6259 50  5 UAAGAGGAAGUAUGCUGUUUU 4062  51  6 AACAGCAGUUGUUGCAGAAUU 5291  52 7 CCAGACAAUAAUUGUCUGGUU 7387  53  8 CCAGACAAUAAUUGUCUGGUU 7387  53  9CCAGACAAUAAUUGUCUGGUU 7387  53 10 CUCCCAGGCUCAGAUCUGGUU   16  54 11CCAGAUCUUCCCUAAAAAAUU 1630  55 12 UUUUUUAUCUGCCUGGGAGUU 7011  56 13UGGGUUCCCUAGUUAGCCAUU   40  57 14 UGGCUAAGAUCUACAGCUGUU 8585  58 15CAGCUGUCCCAAGAACCCAUU 7325  59 16 AUCCUUUGAUGCACACAAUUU  591  60 17AUUGUGUCACUUCCUUCAGUU 6988  61 18 CUGAAGGAAGCUAAAGGAUUU 1785  62 19UCCUGUGUCAGCUGCUGCUUU  685  63 20 AGCAGCAUUGUUAGCUGCUUU 8481  64 21AGCAGCUUUAUACACAGGAUU 9046  65 22 ACCAACAAGGUUUCUGUCAUU 1284  66 23UGACAGAUCUAAUUACUACUU 6573  67 24 GUAGUAAUUAUCUGUUGGUUU 6311  68 25CUGAGGGAAGCUAAAGGAUUU 1785  69 26 AUCCUUUGAUGCACACAAUUU  591  60 27AUUGUGUCACUUCCCUCAGUU 6988 232 28 CAAAGCUAGAUGAAUUGCUUU 3534  70 29AGCAAUUGGUACAAGCAGUUU 5432  71 30 ACUGCUUGUUAGAGCUUUGUU 2952  72 31AGGUCAGGGUCUACUUGUGUU 4872  73 32 CACAAGUGCUGAUAUUUCUUU 5779  74 33AGAAAUAAUUGUCUGACCUUU 7384  75 34 CUAAGUUAUGGAGCCAUAUUU 5212  76 35AUAUGGCCUGAUGUACCAUUU  758  77 36 AUGGUACUUCUGAACUUAGUU 4736  78 37UGGCUCCAUUUCUUGCUCUUU 5365  79 38 AGAGCAACCCCAAAUCCCCUU 7544  80 39GGGGAUUUAGGGGGAGCCAUU 4191  81 40 AUCUCCACAAGUGCUGAUAUU 5784  82 41UAUCAGCAGUUCUUGAAGUUU 8942  83 42 ACUUCAAAUUGUUGGAGAUUU 8158  84 43AGACUGUGACCCACAAUUUUU 5862  85 44 AAAUUGUGGAUGAAUACUGUU 4310  86 45CAGUAUUUGUCUACAGUCUUU  499  87 46 ACAGGCCUGUGUAAUGACUUU 6362  88 47AGUCAUUGGUCUUAAAGGUUU 8559  89 48 ACCUUUAGGACAGGCCUGUUU 6371  90 49UCAGUGUUAUUUGACCCUUUU 6973  91 50 AAGGGUCUGAGGGAUCUCUUU  135  92 51AGAGAUCUUUCCACACUGAUU  158  93 52 CAUAGUGCUUCCUGCUGCUUU 7337  94 53AGCAGCAUUGUUAGCUGCUUU 8481  95 54 AGCAGCUAACAGCACUAUGUU 8190  96 55GCUGCUUAUAUGCAGGAUCUU 9044  97 56 GAUCCUGUCUGAAGGGAUGUU  531  98 57CAUCCCUGUUAAAAGCAGCUU 7118  99 58 UGGUCUAACCAGAGAGACCUU 9081 100 59GGUCUCUUUUAACAUUUGCUU  928 101 60 GCAAAUGUUUUCUAGACCAUU 7557 102 61CUCCCAGGCUCAGAUCUGGUU 9097 103 62 CCAGAUCUUCCCUAAAAAAUU 1630  55 63UUUUUUAUCUGCCUGGGAGUU 7011  56 64 UGGGUUCCCUAGUUAGCCAUU 9121 104 65UGGCUAAGAUCUACAGCUGUU 8585  58 66 CAGCUGUCCCAAGAACCCAUU 7325  59

To Make MV-siRNA complexes targeted to HIV from the sequences in Table 7above, the individual oligos can be combined and annealed as follows.

1) MV-siRNA_1649/1648/1648; Anneal sequences 1 & 2, and 3.2) MV-siRNA_6259/4062/5291; Anneal sequences 4 & 5, and 6.3) MV-siRNA_7387/7387/7387; Anneal sequences 7 & 8, and 9.4) MV-siRNA_16/1630/7011; Anneal sequences 10 & 11, and 12.5) MV-siRNA_40/8585/7325; Anneal sequences 13 & 14, and 15.6) MV-siRNA_591/6988/1785; Anneal sequences 16 & 17, and 18.7) MV-siRNA_685/8481/9046; Anneal sequences 19 & 20, and 21.8) MV-siRNA_1284/6573/6311; Anneal sequences 21 & 22, and 23.9) MV-siRNA_1785/591/6988; Anneal sequences 24 & 25, and 26.10) MV-siRNA_3534/5432/2952; Anneal sequences 27 & 28, and 29.11) MV-siRNA_4872/5779/7384; Anneal sequences 30 & 31, and 32.12) MV-siRNA_5212/758/4736; Anneal sequences 33 & 34, and 35.13) MV-siRNA_5365/7544/4191; Anneal sequences 36 & 37, and 38.14) MV-siRNA_5784/8942/8158; Anneal sequences 39 & 40, and 41.15) MV-siRNA_5862/4310/499; Anneal sequences 42 & 43, and 44.16) MV-siRNA_6362/8559/6371; Anneal sequences 45 & 46, and 47.17) MV-siRNA_6973/135/158; Anneal sequences 48 & 49, and 50.18) MV-siRNA_7337/8481/8190; Anneal sequences 51 & 52, and 53.19) MV-siRNA_9044/531/7118; Anneal sequences 54 & 55, and 56.20) MV-siRNA_9081/928/7557; Anneal sequences 57 & 58, and 59.21) MV-siRNA_9097/1630/7011; Anneal sequences 60 & 61, and 62.22) MV-siRNA.sub_9121/8585/7325; Anneal sequences 63 & 64, and 65.

Example 3 Trivoid Anti-apoB

Multivalent siRNA can be designed to suppress large genes by targetingin 2-3 locations on a single gene. The MV-siRNA can also employalternative RNA chemistries to enhance the Tm during annealing. In thisexample, as shown in Table 8 below, a series of MV-siRNA are designed totarget the apolipoprotein B (ApoB) gene, and the presence of optional2′-O methyl RNA subunits is indicated within parenthesis.

TABLE 8 Trivalent MV-siRNA to ApoB SEQ No. Sequence Target Gene ID NO: 1 (UGGAACU)UUCAGCUUCAUAUU ApoB @ 268 105  2 (UAUGAAG)GCACCAUGAUGUUUApoB @ 9905 106  3 (ACAUCAU)CUUCC(AGUUCCA)UU ApoB @ 1703 107  4(ACUCUUC)AGAGUUCUUGGUUU ApoB @ 448 108  5 (ACCAAGA)CCUUGGAGACACUU ApoB @2288 109  6 (GUGUCUC)AGUUG(GAAGAGU)UU ApoB @ 6609 110  7(ACCUGGA)CAUGGCAGCUGCUU ApoB @ 469 111  8 (GCAGCUG)CAAACUCUUCAGUU ApoB @458 112  9 (CUGAAGA)CGUAU(UCCAGGU)UU ApoB @ 113 12263 10(CAGGGUA)AAGAACAAUUUGUU ApoB @ 520 114 11 (CAAAUUG)CUGUAGACAUUUUU ApoB @4182 115 12 (AAAUGUC)CAGCG(UACCCUG)UU ApoB @ 116 12548 13(CCCUGGA)CACCGCUGGAACUUUU ApoB @ 279 117 14 (AAGUUCC)AAUAACUUUUCCAUUUApoB @ 9161 118 15 (AUGGAAA)AGGCAAG(UCCAGGG)UU ApoB @ 9968 119 16(CCCUGGA)CACCGCUGGAACUUUUU ApoB @ 278 120 17 (AAAGUUC)CAAUAACUUUUCCAUUUApoB @ 9161 121 18 (AUGGAAA)AUGGCAAG(UCCAGGG)UU ApoB @ 9968 122

To make synthetic MV-siRNA trivalent complexes from the sequences inTable 8 above, the individual oligos can be combined and annealed asfollows.

1) MV-siRNA_268/9950/1703; Anneal sequences 1 & 2, and then 3.2) MV-siRNA_448/2288/6609; Anneal sequences 4 & 5, and then 6.3) MV-siRNA_469/458/12263; Anneal sequences 7 & 8, and then 9.4) MV-siRNA_520/4182/12548; Anneal sequences 10 & 11, and then 12.5) MV-siRNA_279/9161/9986; Anneal sequences 13 & 14, and then 15.6) MV-siRNA_278/9161/9986; Anneal sequences 16 & 17, and then 18.

Multivalent siRNA that are designed with potent primary and secondarystrands can also employ wobble or universal bases to complete targetcomplimentarity, or blunt ended DNA to deactivate the strand fromsilencing any target. Exemplary oligos directed to ApoB are shown inTable 9 below, in which (*) indicates an optional wobble or universalbase.

TABLE 9 Exemplary Bivalent MV-siRNA to ApoB No. Sequence Target GeneSEQ ID NO: 19 UGAAUCGAGUUGCAUCUUUUU ApoB @ 223 123 20AAAGAUGCUGCUCAUCACAUU ApoB @ 883 124 21 UGUGAUGACACUCGAUUCAUU ApoB @10116 (G/A pairs) 125 22 U*UGAU*ACACUCGAUUCAUU ApoB @ 10116 (univ. base)126 23 TGTGATGACACTCGATTCA null @ 10166 127 24 CAGCUUGAGUUCGUACCUGUUApoB @ 483 128 25 CAGGUACAGAGAACUCCAAUU ApoB @ 11596 129 26UUGGAGUCUGACCAAGCUGUU ApoB @ 2454 130 27 UUGGAGUCUGAC*AAGCU*UU ApoB @2454 131 28 TTGGAGTCTGACCAAGCTG null @ 2454 132

To make synthetic MV-siRNA bivalent complexes from the sequences inTable 9 above, the individual oligos can be combined and annealed asfollows.

7a) MV-siRNA_223/883/10116); Anneal sequences 19, 20, and 21.7b) MV-siRNA_223/883/10116*); Anneal sequences 19, 20, and 22.7c) MV-siRNA_223/883/null); Anneal sequences 19, 20, and 23.8a) MV-siRNA_483/11596/2454); Anneal sequences 24, 25, and 26.8b) MV-siRNA_483/11596/2454*); Anneal sequences 24, 25, and 26.8c) MV-siRNA_483/11596/null); Anneal sequences 24, 25, and 26.

Multivalent-siRNAs can also be designed to suppress large genes bytargeting 2-3 locations on a single gene. As noted, above, certainembodiments of the instant MV-siRNAs can also employ alternative RNAchemistries to enhance the Tm during annealing. In Table 10 below,optional 2′-O methyl RNA 2′-fluoro bases are indicated withinparenthesis. Among other examples of alternate bases, 5-methyl can alsoincrease Tm of MV-siRNA structure, if desired.

TABLE 10 Exemplary Trivalent MV-siRNA to ApoB  No. Sequence Target GeneSEQ ID NO:  1 UGG(AA)CUUUCAGCUUCAUAUU ApoB @ 268 105  2U(AU)GAAGGCACCAUGAUGUUU ApoB @ 9905 106  3 (ACAUCAU)CUUCCAGUUCCAUUApoB @ 1703 107  4 AC(U)CUUCAGAGUUCUUGGUUU ApoB @ 448 108  5(ACCAAGA)CCUUGGAGACACUU ApoB @ 2288 109  6 G(U)GUCUCAGUUGGAAGAGUUUApoB @ 6609 110  7 (ACCUGGA)CAUGGCAGCUGCUU ApoB @ 469 111  8GC(A)GCUGCAAACUCUUCAGUU ApoB @ 458 112  9 (CUGAAGA)CGUAU(UCCAGGU)UUApoB @ 12263 113 10 (CAGGGUA)AAGAACAAUUUGUU ApoB @ 520 114 11(CAAAUU)GCUGUAGACA(UUU)UU ApoB @ 4182 115 12 (AAAUGUC)CAGCGUACCCUGUUApoB @ 12548 116 13 (CCCUGGA)CACCGCUGGAACUUUU ApoB @ 279 117 14(AAGUUCC)AAUAACUUUUCCAUUU ApoB @ 9161 118 15 (AU)GGAAAAGGCAAG(UCCAGGG)UUApoB @ 9968 119 16 CCC(U)GGACACCGCUGG(AACUUU)UU ApoB @ 278 120 17(AAA)GUUCCAAUAACUU(UU)CC(AU)UU ApoB @ 9161 121 18(AUGGAAA)AUGGCAAG(UCCAGGG)UU ApoB @ 9968 122 19 UCAGGGCCGCUCUGUAUUUUUApoB @ 6427 133 20 AAAUACAUUUCUGGAAGAGUU ApoB @ 8144 134 21CUCUUCCAAAAAGCCCUGAUU ApoB @ 12831 135 22AAAUACAUUUCUGGAAGAGuu&CUCUUCCAAAAA Linker construct for 136GCCCUGAuu&UCAGGGCCGCUCUGUAUUUuu cleavage after annealing. “&” = PCSpacer, or linkage phosphoramidite

To make synthetic MV-siRNA bivalent complexes from the sequences inTable 10 above, the individual oligos can be combined and annealed asfollows.

1) MV-siRNA_268/9950/1703; Anneal sequences 1 & 2, and then 3.2) MV-siRNA_448/2288/6609; Anneal sequences 4 & 5, and then 6.3) MV-siRNA_469/458/12263; Anneal sequences 7 & 8, and then 9.4) MV-siRNA_520/4182/12548; Anneal sequences 10 & 11, and then 12.5) MV-siRNA_279/9161/9986; Anneal sequences 13 & 14, and then 15.6) MV-siRNA_278/9161/9986; Anneal sequences 16 & 17, and then 18.7) MV-siRNA_6427/8144/12831; Anneal sequences 19 & 20, and then 21.7b) MV-siRNA_6427/8144/12831; Anneal strand 22, then cleave linkagephosphate with ammonium hydroxide. 7b) MV-siRNA_6427/8144/12831; Annealstrand 22, then cleave PC Spacer with UV light in the 300-350 nmspectral range.

In certain embodiments, multivalent-siRNA that are designed with potentprimary and secondary strands can employ wobble, spacer, or abasic basetypes (examples are indicated by (*) in Table 11 below) to completetarget compliments, or blunt ended DNA to deactivate the strand fromsilencing any target. In some embodiments, UNA, linker phosphoramidites,rSpacer, 5-nitroindole can act as effective abasic bases in place ofmismatched nucleotides. If desired, the use of abasic bases can resultin weakened Tm, and/or pyrimidines surrounding an abasic site canutilize 2′-fluoro bases to increase Tm by about 2 degrees for every2′-fluoro base.

TABLE 11 Exemplary MV-siRNA Targeted to ApoB  No. Sequence Target GeneSEQ ID NO: 23 UGAAUCGAGUUGCAUCUUUUU ApoB @ 223 123 24AAAGAUGCUGCUCAUCACAUU ApoB @ 883 124 25 UGUGAUGACACUCGAUUCAUU ApoB @10116 (G/A pairs) 125 26 U*UGAU*ACACUGAUUCAUU ApoB @10116 (* rSPACER base) 126 27 TGTGATGACACTCGATTCA null @ 10116 127 28CAGCUUGAGUUCGUACCUGUU ApoB @ 483 128 29 CAGGUACAGAGAACUCCAAUU ApoB @11596 129 30 UUGGAGUCUGACCAAGCUGUU ApoB @ 2454 130 31UUGGAGUCUGAC*AAGCU*UU ApoB @ 2454 (* abasic base) 131 32TTGGAGTCTGACCAAGCTG null @ 2454 132 33 AACCCACUUUCAAAUUUCCUU ApoB @ 9244137 34 GGAAAUUGAGAAUUCUCCAUU ApoB @ 1958 138 35 UGGAGAAUCUCAGUGGGUUUUApoB @ 8005 139 36 rUrGrGfA-fArArUrCrUrCrA-fUrGrGrG-fUrUrU ApoB @ 8005140 37 GAUGAUGAAACAGUGGGUUUU ApoB @ 10439 141 38 AACCCACUUUCAAAUUUCCUUApoB @ 9244 137 39 GGAAAUUGGAGACAUCAUCUU ApoB @ 2284 142 40-rGfAfAfArUrUrGrGrArGrArCfA-rCfArUrCrUrU ApoB @ 2284 143 41GCAAACUCUUCAGAGUUCUUU ApoB @ 452 144 42 AGAACUCCAAGGGUGGGAUUU ApoB @11588 145 43 AUCCCACUUUCAAGUUUGCUU ApoB @ 9244 146 44fA-rCrCrCrArCrUrUrUrCrAfA-fUrUrU-rC ApoB @ 9244 147

To make synthetic MV-siRNA bivalent complexes from the sequences inTable 11 above, the individual oligos can be combined and annealed asfollows.

7a) MV-siRNA_223/883/10116); Anneal sequences 23, 24, and 25.7b) MV-siRNA_223/883/10116*); Anneal sequences 23, 24, and 26.7c) MV-siRNA_223/883/null); Anneal sequences 23, 24, and 27.8a) MV-siRNA_483/11596/2454); Anneal sequences 28, 29, and 30.8b) MV-siRNA_483/11596/2454*); Anneal sequences 28, 29, and 31.8c) MV-siRNA_483/11596/null); Anneal sequences 28, 29, and 32.9) MV-siRNA_9244/1958/8005); Anneal sequences 33, 34, and 35.9b) MV-siRNA_9244/1958/8005); Anneal sequences 33, 34, and 36.10) MV-siRNA_10439/9244/2284); Anneal sequences 37, 38, and 39.10b) MV-siRNA_10439/9244/2284); Anneal sequences 37, 38, and 40.11) MV-siRNA_452/11588/9244); Anneal sequences 41, 42, and 43.11b) MV-siRNA_452/11588/9244); Anneal sequences 41, 42, and 44.

As exemplified in Table 12 below, multivalent siRNA can be targetedagainst human ApoB. Bivalent MV-siRNA can function with varioustolerances to structure and target complementarity of each strand

TABLE 12 Exemplary Multivalent-siRNA  Targeted to Human ApoB ApoB GeneSEQ No. Sequence site ID NO:  1 CUUCAUCACUGAGGCCUCUUU  1192 148  2AGAGGCCAAGCUCUGCAUUUU  5140 149  3 AAUGCAGAUGAAGAUGAAGAA 10229 150  4UUCAGCCUGCAUGUUGGCUUU  2724 151  5 AGCCAACUAUACUUGGAUCUU 13294 152  6GAUCCAAAAGCAGGCUGAAGA  4960 153  7 CCCUCAUCUGAGAAUCUGGUU  8927 154  8CCAGAUUCAUAAACCAAGUUU  9044 155  9 ACUUGGUGGCCCAUGAGGGUU  3440 156 10UCAAGAAUUCCUUCAAGCCUU  9595 157 11 GGCUUGAAGCGAUCACACUUU   758 158 12AGUGUGAACGUAUUCUUGAUU  4367 159 13 UUGCAGUUGAUCCUGGUGGUU   344 160 14CCACCAGGUAGGUGACCACUU  1354 161 15 GUGGUCAGGAGAACUGCAAUU  2483 162 16CCUCCAGCUCAACCUUGCAUU   358 163 17 UGCAAGGUCUCAAAAAAUGUU  6341 164 18CAUUUUUGAUCUCUGGAGGUU  4043 165 19 CAGGAUGUAAGUAGGUUCAUU   570 166 20UGAACCUUAGCAACAGUGUUU  5687 167 21 ACACUGUGCCCACAUCCUGUU  9109 168 22GGCUUGAAGCGAUCACACUUU   758 169 23 AGUGUGAACGUAUUCUUGUUU  4367 170 24ACAAGAAUUCCUUCAAGCCUU  9595 171 25 UGAAGAGAUUAGCUCUCUGUU  1153 172 26CAGAGAGGCCAAGCUCUGCUU  5143 173 27 GCAGAGCUGGCUCUCUUCAUU 10304 174 28CUCAGUAACCAGCUUAUUGUU  1170 175 29 CAAUAAGAUUUAUAACAAAUU  7084 176 30UUUGUUAUCUUAUACUGAGUU  9650 177 31 GAACCAAGGCUUGUAAAGUUU  1258 178 32ACUUUACAAAAGCAACAAUUU  6286 179 33 AUUGUUGUUAAAUUGGUUCUU  6078 180 34CAGGUAGGUGACCACAUCUUU  1350 181 35 AGAUGUGACUGCUUCAUCAUU  1203 182 36UGAUGAACUGCGCUACCUGUU  8486 183 37 CCAGUCGCUUAUCUCCCGGUU  1786 184 38CCGGGAGCAAUGACUCCAGUU  2678 185 39 CUGGAGUCAUGGCGACUGGUU  2486 186 40UGGAAGAGAAACAGAUUUGUU  2046 187 41 CAAAUCUUUAAUCAGCUUCUU  2403 188 42GAAGCUGCCUCUUCUUCCAUU 12299 189 43 AUCCAAAGGCAGUGAGGGUUU  2152 190 44ACCCUCAACUCAGUUUUGAUU 12242 191 45 UCAAAACCGGAAUUUGGAUUU  3316 192 46UAGAGACACCAUCAGGAACUU  2302 193 47 GUUCCUGGAGAGUCUUCAAUU  1102 194 48UUGAAGAAUUAGGUCUCUAUU  1153 195 49 GCUCAUGUUUAUCAUCUUUUU  2350 196 50AAAGAUGCUGAACUUAAAGUU  7622 197 51 CUUUAAGGGCAACAUGAGCUU  2863 198 52GGAGCAAUGACUCCAGAUGUU  2675 199 53 CAUCUGGGGGAUCCCCUGCUU  2544 200 54GCAGGGGAGGUGUUGCUCCUU   912 201 55 UCACAAACUCCACAGACACUU  2761 202 56GUGUCUGCUUUAUAGCUUGUU  5672 203 57 CAAGCUAAAGGAUUUGUGAUU  9683 204 58GCAGCUUGACUGGUCUCUUUU  2914 205 59 AAGAGACUCUGAACUGCCCUU  4588 206 60GGGCAGUGAUGGAAGCUGCUU  8494 207 61 CAGGACUGCCUGUUCUCAAUU  2996 208 62UUGAGAACUUCUAAUUUGGUU  8522 209 63 CCAAAUUUGAAAAGUCCUGUU  9855 210 64UGUAGGCCUCAGUUCCAGCUU  3132 211 65 GCUGGAAUUCUGGUAUGUGUU  8335 212 66CACAUACCGAAUGCCUACAUU  9926 213 67 GACUUCACUGGACAAGGUCUU  3300 214 68GACCUUGAAGUUGAAAAUGUU  5301 215 69 CAUUUUCUGCACUGAAGUCUU 11983 216 70AAGCAGUUUGGCAGGCGACUU  3549 217 71 GUCGCCUUGUGAGCACCACUU  5039 218 72GUGGUGCCACUGACUGCUUUU 12521 219 73 CAGAUGAGUCCAUUUGGAGUU  3568 220 74CUCCAAACAGUGCCAUGCCUU  9142 221 75 GGCAUGGAGCCUUCAUCUGUU  3256 222 76CACAGACUUGAAGUGGAGGUU  4086 223 77 CCUCCACUGAGCAGCUUGAUU  2924 224 78UCAAGCUUCAAAGUCUGUGUU   974 225 79 AUGGCAGAUGGAAUCCCACUU  4102 226 80GUGGGAUCACCUCCGUUUUUU  2971 227 81 AAAACGGUUUCUCUGCCAUUU 12836 228 82UGAUACAACUUGGGAAUGGUU  4148 229 83 CCAUUCCCUAUGUCAGCAUUU  2971 230 84AUGCUGACAAAUUGUAUCAUU 12836 231

To make synthetic MV-siRNA bivalent complexes from the sequences inTable 12 above, the individual oligos can be combined and annealed asfollows. MV-siRNA; Anneal sequences 1, 2, and 3. MV-siRNA; Annealsequences 4, 5, and 6. MV-siRNA; Anneal sequences 7, 8, and 9. MV-siRNA;Anneal sequences 10, 11, and 12. MV-siRNA; Anneal sequences 13, 14, and15. MV-siRNA; Anneal sequences 16, 17, and 18. MV-siRNA; Annealsequences 19, 20, and 21. MV-siRNA; Anneal sequences 22, 23, and 24.MV-siRNA; Anneal sequences 25, 26, and 27. MV-siRNA; Anneal sequences28, 29, and 30. MV-siRNA; Anneal sequences 31, 32, and 33. MV-siRNA;Anneal sequences 34, 35, and 36. MV-siRNA; Anneal sequences 37, 38, and39. MV-siRNA; Anneal sequences 40, 41, and 42. MV-siRNA; Annealsequences 43, 44, and 45. MV-siRNA; Anneal sequences 46, 47, and 48.MV-siRNA; Anneal sequences 49, 50, and 51. MV-siRNA; Anneal sequences52, 53, and 54. MV-siRNA; Anneal sequences 55, 56, and 57. MV-siRNA;Anneal sequences 58, 59, and 60. MV-siRNA; Anneal sequences 61, 62, and63. MV-siRNA; Anneal sequences 64, 65 and 66. MV-siRNA; Anneal sequences67, 68, and 69. MV-siRNA; Anneal sequences 70, 71, and 72. MV-siRNA;Anneal sequences 73, 74, and 75. MV-siRNA; Anneal sequences 76, 77, and78. MV-siRNA; Anneal sequences 79, 80, and 81. MV-siRNA; Annealsequences 82, 83, and 84.

MV-siRNA directed to ApoB can be used to treat or manage a wide varietyof diseases or conditions associated with the expression of that targetprotein, as described herein and known in the art.

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1. A polynucleotide complex of at least three separate polynucleotides,comprising (a) a first polynucleotide comprising a target-specificregion that is complementary to a first target sequence, a 5′ region,and a 3′ region; (b) a second polynucleotide comprising atarget-specific region that is complementary to a second targetsequence, a 5′ region, and a 3′ region; and (c) a third polynucleotidecomprising a null region or a target-specific region that iscomplementary to a third target specific, a 5′ region, and a 3′ region,wherein each of the target-specific regions of the first, second, andthird polynucleotides are complementary to a different target sequence,wherein the 5′ region of the first polynucleotide is complementary tothe 3′ region of the third polynucleotide, wherein the 3′ region of thefirst polynucleotide is complementary to the 5′ region of the secondpolynucleotide, and wherein the 3′ region of the second polynucleotideis complementary to the 5′ region of the third polynucleotide, andwherein the three separate polynucleotides hybridize via theircomplementary 3′ and 5′ regions to form a polynucleotide complex with afirst, second, and third single-stranded region, and a first, second,and third self-complementary region.
 2. The polynucleotide complex ofclaim 1, wherein the first, second, and/or third polynucleotidecomprises about 15-30 nucleotides.
 3. The polynucleotide complex ofclaim 1, wherein the first, second, and/or third polynucleotidecomprises about 17-25 nucleotides.
 4. The polynucleotide complex ofclaim 1, wherein one or more of the self-complementary regions comprisesabout 5-10 nucleotide pairs.
 5. The polynucleotide complex of claim 1,wherein one or more of the self-complementary regions comprises about7-8 nucleotide pairs.
 6. The polynucleotide complex of claim 1, whereineach of said first, second, and third target sequences are present inthe same gene, cDNA, mRNA, or microRNA.
 7. The polynucleotide complex ofclaim 1, wherein at least two of said first, second, and third targetsequences are present in different genes, cDNAs, mRNAs, or microRNAs. 8.The polynucleotide complex of claim 1, wherein all or a portion of the5′ and/or 3′ region of each polynucleotide is also complementary to thetarget sequence for that polynucleotide.
 9. The polynucleotide complexof claim 1, wherein one or more of the self-complementary regionscomprises a 3′ overhang.
 10. A self-hybridizing polynucleotide molecule,comprising (a) a first nucleotide sequence comprising a target-specificregion that is complementary to a first target sequence, a 5′ region,and a 3′ region, (b) a second nucleotide sequence comprising atarget-specific region that is complementary to a second targetsequence, a 5′ region, and a 3′ region; and (c) a third nucleotidesequence comprising a null region or a target-specific region that iscomplementary to a third target sequence, a 5′ region, and a 3′ region,wherein the target-specific regions of each of the first, second, andthird nucleotide sequences are complementary to a different targetsequence, wherein the 5′ region of the first nucleotide sequence iscomplementary to the 3′ region of the third nucleotide sequence, whereinthe 3′ region of the first nucleotide sequence is complementary to the5′ region of the second nucleotide sequence, and wherein the 3′ regionof the second nucleotide sequence is complementary to the 5′ region ofthe third nucleotide sequence, and wherein each of the 5′ regionshybridizes to their complementary 3′ regions to form a self-hybridizingpolynucleotide molecule with a first, second, and third single-strandedregion, and a first, second, and third self-complementary region. 11.The self-hybridizing polynucleotide molecule of claim 10, wherein thefirst, second, or third nucleotide sequence comprises about 15-60nucleotides.
 12. The self-hybridizing polynucleotide molecule of claim10, wherein the target-specific regions each comprise about 15-30nucleotides.
 13. The self-hybridizing polynucleotide molecule of claim10, wherein one or more of the self-complementary regions comprisesabout 10-54 nucleotides.
 14. The self-hybridizing polynucleotidemolecule of claim 10, wherein one or more of the self-complementaryregions comprises a 3′ overhang.
 15. The self-hybridizing polynucleotidemolecule of claim 10, wherein one or more of the self-complementaryregions forms a stem-loop structure.
 16. The self-hybridizingpolynucleotide molecule of claim 10, wherein one or more of theself-complementary regions comprises a proximal box of dinucleotidesAG/UU that is outside of the target specific region
 17. Theself-hybridizing polynucleotide molecule of claim 10, wherein one ormore of the self-complementary regions comprises a distal box of 4nucleotides that is outside of the target-specific region, wherein thethird nucleotide of the distal box is not a G.
 18. The self-hybridizingpolynucleotide molecule of claim 10, wherein each of said first, second,and third target sequences are present in the same gene, cDNA, mRNA, ormicroRNA.
 19. The self-hybridizing polynucleotide molecule of claim 10,wherein at least two of said first, second, and third target sequencesare present in different genes, cDNAs, mRNAs, or microRNAs.
 20. A vectorthat encodes a self-hybridizing polynucleotide molecule according toclaim
 10. 21-25. (canceled)